Acute sulphide toxicity in Perinereis aibuhitensis under different salinities and temperatures: LC50 and antioxidant responses

Sulphide accumulates in sediment due to global aquaculture development and is very harmful to aquatic and benthic ecosystems. Perinereis aibuhitensis inhabits coastal areas and is often exposed to fairly high sulphide concentrations. The present study investigated the tolerance of P. aibuhitensis to sulphide to evaluate its potential application in sulphide remediation and sustainable aquaculture. We assessed the toxicity of sulphide on P. aibuhitensis in a 96 h acute sulphide exposure experiment under different temperature and salinity conditions. Two-way ANOVA showed that increasing salinity did not influence the LC50 of P. aibuhitensis exposed to sulphide. In contrast, increasing temperature significantly augmented the LC50 value (p < 0.05). The results showed a negative relationship between mortality and temperature, and between mortality and exposure time. Subsequently, we performed 2-way ANOVA analysis of the antioxidant (superoxide dismutase [SOD], catalase [CAT] and total antioxidant capacity [T-AOC]) responses of P. aibuhitensis during 96 h exposure to sub-lethal sulphide concentrations (0, 80, 160, 320, and 640 μmol l−1), and a sub sequent recovery period. The activation of this antioxidant defense system appeared to depend on sulphide concentration and exposure duration and their interaction. SOD, CAT, and T-AOC showed obvious differences at the beginning and end of exposure. They were steadily restored during the recovery period. The results indicated that P. aibuhitensis adjusts its antioxidant defense system to cope with sulphide contamination. Therefore, these indexes of P. aibuhitensis could be applied to environmental monitoring and bio-restoration at mudflat or intensive aquaculture areas with high sulphide concentrations.


INTRODUCTION
Since intensive aquaculture has increased rapidly, concerns have grown about the effects of contaminants (e.g.organic enrichment, sulphide, biogenic elements) derived from aquaculture farming on the biogeochemical processes of benthic sediment, espe-cially in coastal cultivation zones (Grant et al. 1995, Holmer et al. 2005, Wilson & Vopel 2015).Increased deposition of organic matter changes sediment biogeochemistry, which can result in oxygen depletion (Holmer et al. 2005, Metzger et al. 2014).A low oxygen concentration in the benthic environment promotes anaerobic metabolism, sulphate reduction and consequently a higher concentration of dissolved sulphide in sediment pore water (Newell 2004, Sakai et al. 2013).Sulphide (the sum of H 2 S, HS − , and S 2− ) is known to be extremely toxic to most aerobic life at nanomolar to micromolar concentrations (Lamers et al. 2013).It is impossible to prevent sulphide from entering the body of aquatic organisms, and benthic organisms have evolved abilities to resist the toxicity of hydrogen sulphide (Harald & Marianne 2015).
Sulphide has deleterious effects on aqua culture environments and on benthic macrofauna (Riedel et al. 2012, Kilminster et al. 2014).It can cause hypoxia in tissues (Hauschild & Gries haber 1997) and can bind at more than one enzyme site to inhibit cytochrome c oxidase and many other enzymes, thereby inhibiting the aerobic metabolism and enzyme activity (Vismann 1990, Völkel & Grieshaber 1994, Jimenez-Gutierrez et al. 2014).However, many benthic animals tolerate the toxicity of sulphide through special eco-physiological strategies (Brouwer & Murphy 1995, Hildebrandt & Grieshaber 2008).Early studies showed that the sulphide in pore water affects the distribution of the nereid polychaetes.Nereis virens is found in areas with low sulphide levels (< 50 µM), while Nereis succinea is distributed in sediments with high sulphide levels (50−2000 µM) and Nereis diversicolor has a broader tolerance with regard to sulphide in porewater and is more tolerant to sulphide than N. virens (Kristensen 1988).Burrowing, macrofauna such as polychaetes enhance oxic conditions through bioturbation and bioirrigation (Goldhaber et al. 1977, Carvalho et al. 2007), which may diminish the possible negative effects of sulphide in sediment.
Aquatic organisms possess antioxidant enzymes to protect their cellular systems from oxidative damage induced by xenobiotics that can cause stresses through induction of a disbalance between the generation and elimination of reactive oxygen species (ROS) (Regoli & Giuliani 2014, González et al. 2015).Studies have indicated that when marine organisms are exposed to oxidative stress, the activity of antioxidant enzymes will change with pollutant concentration and exposure time (Vidal-Liñán et al. 2010, Qiu et al. 2013).The fluctuations in activity of antioxidant en zymes, such as superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) in polychaetes exposed to sulphide could help us to understand its physiological responses.
Polychaetes play an essential role in coastal benthic ecosystems (Leung & Cheung 2017, Weis et al. 2017).They are important sources of food for fish and other benthic species (Fadhullah & Syakir 2016).
They also play a role in controlling algal blooms, bioaccumulate heavy metals, and enhance oxygen and nutrient transformation at the sediment−water interface through their deposit and suspension feeding and reworking activity in coastal sediments (Baumann & Fisher 2011, Leung & Cheung 2017, Weis et al. 2017).Perinereis aibuhitensis (Grube) is an im portant commercial polychaete and is widespread in China.P. aibuhitensis prefers water with salinities between 24 and 32 and its respiration increases with increasing temperature within the range 10− 27°C (Wang et al. 2004, Cai & Yan 2014).It has been shown that P. aibuhitensis can be used as a remediation species co-cultured in shrimp or fish farms to convert wastes to valuable biomass (Honda & Kikuchi 2002, Fang et al. 2014, 2016).In addition, it is an indicator for multiple-contaminant (e.g.heavy metals and petroleum hydro carbons) accumulation and remediation (Ge et al. 2016a,b).Deng et al. (2006) found that the accumulation of sulphide in sediment was reduced when P. aibuhitensis was introduced into shrimp ponds.
The objectives of this study were to evaluate the tolerance of the polychaete P. aibuhitensis to different sulphide concentrations and its recovery ability after being removed from sulphide.We also evaluated the activity of its antioxidant systems during sulphide exposure and a subsequent recovery period.The results will be helpful for evaluating the application of polychaetes in bio-monitoring and biorestoration of sulphide-contaminated areas.

Animals and general conditions
Perinereis aibuhitensis were collected from Dingzi Bay, a semi-enclosed bay in southeastern Shandong Province, PR China, in September 2015.Polychaetes were transported to the laboratory in wet sawdust and maintained in a recirculating system filled with filtered seawater (salinity 30, pH 7.8−8.1,temperature 18°C) containing 10 cm of sand before the experiments.Organisms were fed with a mixture of fish fodder, algae powder, wheat bran, and dia toms.A simulated natural photoperiod (14 h light: 10 h dark) was used throughout the experiment.During the day the light intensity was less than 100 lux to prevent photolysis of sulphide, except for the short period during which the samples were collected.Animals were acclimated in the recirculation tanks for 7 d before the study.

Acute toxicity test
A 96 h assay was conducted to evaluate the toxicity of sulphide on P. aibuhitensis.A stock solution of 160 mmol l −1 sulphide (sum of H 2 S, HS − , and S 2− ) was prepared by mixing the Na 2 S•9H 2 O crystals with N 2saturated distilled water.Then, the stock solution was adjusted to pH 8.0 with 1 mol l −1 HCl.The experimental setup was a flow-through system consisting of a stock solution tank, a filtered seawater tank, and an incubation chamber (Fig. 1), which kept both dissolved oxygen (DO) and H 2 S at a constant level.One system can connect with 1−8 incubation chambers in which one grid can hold 2 polychaetes.The flow rate of the stock solution and seawater were adjusted to create different sulphide concentrations.Stock solution and seawater were mixed uniformly by a small pump before entering the incubation chamber.A stock solution tank was placed in darkness and sealed with paraffin oil to maintain the solution concentration.The stock solution was re-filled at intervals of 6−8 h.The time for the mixed seawater going through the incubation chamber was about 10−15 min.
Intact and healthy polychaetes (1.38 ± 0.12 g, mean ± SD) were selected for the experiment.Experiments were carried out by varying 3 factors: sulphide concentration, salinity, and temperature.Experimental concentrations of sulphide were 0, 80, 160, 320, 640, 1280, and 2560 µM l −1 , and the corresponding hydrogen sulphide (H 2 S) concentrations (Unisense) in the incu bation chamber were 0, 10. 34, 22.44, 41.31, 53.09, 91.62, and 148.21 µM l −1 , respectively.The salinities were 30, 27, and 24, and the temperatures were 25, 20, and 15°C.In total, there were 63 treatments with 4 replicates of each.Ten individuals were placed in each replicate, and a total of 2520 polychaetes were used.There were 21 systems with 4 incubation chambers for each temperature treatment at one time.The process above was conducted for one salinity at a time.The DO level was above 5.12 mg l −1 .The salinities and temperatures were ad justed gradually by 1−2 units per day before the 7 d acclimation period.

Sample collection and preparation of tissue extracts
Three hundred polychaetes were collected randomly after acclimation to laboratory temperature in the recirculating tanks.Then they were exposed to a series of sublethal sulphide concentrations (0, 80, 160, 320, and 640 µM l −1 ) for 96 h.These sulphide concentrations were lower than the calculated LC 50 value at 15°C.In the recovery experiments, sulphide toxicity was removed and all animals were then placed in clean seawater for 96 h.There were 4 replicates in all treatments (n = 15).The water temperature was maintained at 15°C, salinity was 30, and pH was 8.0.Samples were collected after 6, 12, 24, 48, 72, and 96 h during both the sulphide exposure and sulphide recovery periods for determination of Fig. 1.The flow-through system used in this study enzyme activity.Polychaetes were dissected from between the head and the beginning of the tail, as there are no morphological changes in this region along its body length (Wohlgemuth et al. 2000).Tissue samples were placed in centrifuge tubes, immediately frozen in liquid nitrogen, and then stored at −80°C until analysis.
Tissue samples were homogenized individually in 9 volumes (the ratio of buffer volume to tissue weight) of PBS buffer solution (pH 7.2−7.8)with multifunctional homogenizer (Precellys 24 Dual) at 4°C.Homo genates were then centrifuged at 2016 × g for 10 min at 4°C.Supernatants for the determination of SOD, CAT, and T-AOC were tested within 12 h.SOD, CAT, and T-AOC were analyzed using kits (Nanjing Jiancheng Bioengineering Institute) based on the Coomassie Blue protein assay.
Enzyme activity test SOD activity was determined by the ability of this enzyme to inhibit the reduction of nitro blue tetrazolium at 37°C (Wu & Tiedemann 2002).CAT decomposes hydrogen peroxide, thereby preventing its accumulation.This reaction can be ceased by ammonium molybdate, producing a yellow complex compound alongside hydrogen peroxide, which can be determined by absorbance at 405 nm (Sánchez-Valle et al. 2012, Tian et al. 2014).T-AOC was determined by its ability to reduce Fe 3+ to Fe 2+ , which was measured using the absorbance at 520 nm, where 1 unit is defined an increase in optical density (OD) of 0.01 per minute (He et al. 2013).Protein content was detected using the Coomassie Blue staining method (Brunelle & Green 2014).The enzyme activities are expressed as U mg protein −1 .

Statistical analysis
All treatments were carried out with 4 replicates.The data are expressed as means ± SD.LC 50 (concentration which causes 50% mortality) values of H 2 S were calculated by linear interpolation.The safe concentration (SC) was calculated based on the following formula: SC = 96 × LC 50 × 0.1.Two-way ANOVA (main effect) was used to identify the effect of salinity and temperature on LC 50 of H 2 S at each sampling time.The interactions between sulphide concentration and exposure and recovery time on the enzyme activities of the polychaetes were also tested by 2way ANOVA (SPSS 13.0 for Windows).If an interaction between the 2 main factors in the 2-way ANOVA model was significant, one-way ANOVA was used to examine the effects of sulphide concentrations on the enzyme activities at a given exposure time.Homogeneity of variances was tested with a Levene test.If the homogeneity of variances was not significant, a Bonferroni test was used during multiple comparisons.Otherwise, the non-parametric Dunnett's T3 test was used.The statistical significance level is p < 0.05.

Acute toxicity and LC 50
During the 96 h toxicity test period, no mortality of polychaetes was observed in the control solutions at any salinity and temperature.The LC 50 and SC values of H 2 S are shown in Table 1.The 2-way ANOVA (main effect) showed that there was no significant difference among the different salinity treatments (24−30) ( (p < 0.05, Fig. 2).When the polychaetes were exposed to over 1280 µM l −1 of H 2 S, LC 50 was reached reached after 72 h at 15°C, after 48 h exposure at 20°C in treatments.All animals died after 72 h exposure in all treatments at 25°C.Morphological abnormalities of the dead polychaetes included the phenomena of curled tail, swollen numb head, dark body color, and occasionally rupture of the epidermis.A polychaete was determined dead when it failed to respond to touch by a glass rod (Llansó 1991).

Sulphide exposure and recovery experiment
In the sulphide exposure and recovery experiment, only 2 polychaetes died and both were in the 640 µM l −1 treatment.

SOD activity
The SOD activity of Perinereis aibu hitensis under sulphide stress is shown in Fig. 3. Two-way ANOVA showed that SOD activity was significantly affected by sulphide concentration (p < 0.05), exposure and recovery time (p < 0.05), and their interaction (p < 0.05, Table 3).Post-hoc tests showed that SOD activity at all concentrations of sulphide was lower than the control after short-term (< 6 h) exposure.It was significantly lower in the 320 µM l −1 treatment than the control (p < 0.05).However, the 320 µM l −1 treatment showed significantly higher SOD activity than those of the other treatments after 48 h (p < 0.05).SOD activity in creased at all concentrations at 96 h compared with the control, which was significant at the higher concentrations (320 and 640 µM l −1 ) (p < 0.05).After releasing the polychaetes from sulphide for 6 h, there was no significant difference in SOD activity among all treatments.However, it increased greatly in the 640 µM l −1 treatment compared with the control after the polychaete had been removed from sulphide for 12 h (p < 0.05), while it was lower than the control in the 80 and 320 µM l −1 treatments (p < 0.05).SOD activity decreased significantly in the lower concentration (80 and 160 µM l −1 ) treatments (p < 0.05) after recovery for 48 h.Furthermore, there were significantly low SOD activities in all treatments at 72 h of recovery (p < 0.05).SOD activity remained low in the 320 µM l −1 treatment at 96 h of recovery.

CAT activity
Two-way ANOVA showed that the CAT activity was significantly affected by sulphide concentration (p < 0.05), exposure and recovery time (p < 0.05), and their interaction (p < 0.05, Table 3, Fig. 4).Subsequently, post-hoc tests showed that the activity of CAT increased significantly in the lower concentration treatments (80 and 160 µM l −1 ) during the initial 6 h sulphide-stress period (p < 0.05).At 12 h, the CAT activity increased greatly relative to the control (p < 0.05) and was positively correlated with sulphide.The CAT activity in P. aibuhitensis decreased in the 80 µM l −1 treatment and increased in the higher concentration treatments (320 and 640 µM l −1 ) after exposure to sulphide for 24 h.The 80, 160 and 640 µM l −1 treatments exhibited lower CAT activity compared with the control at 48 h (p < 0.05).The CAT activity in all of the treatments was reduced relative to the control after 96 h of exposure (p < 0.05).The CAT activity re mained low in the sulphide-stressed groups compared  with the control at the beginning of the recovery period (Fig. 4).There was significantly high CAT activity in the 160 µM l −1 treatment after 12 h of recovery (p < 0.05), but CAT activity of the 160 and 640 µM l −1 treatments decreased significantly at 72 h of recovery from sulphide (p < 0.05).Furthermore, significantly low CAT activity was observed in all treatments after 96 h of recovery (p < 0.05).

T-AOC
T-AOC in P. aibuhitensis exposed to sulphide is shown in Fig. 5.As with SOD and CAT activity, the 2way ANOVA showed T-AOC was also significantly affected by sulphide concentration (p < 0.05) and ex-posure and recovery time (p < 0.05).Moreover, there was a significant interaction be tween the 2 factors (p < 0.05, Table 3).Subsequently, post-hoc tests showed that at the initial 6 h exposure to sulphide, T-AOC in all treatments increased relative to the control.During 12−48 h exposure, T-AOC under different sulphide concentrations were similar to that of the control, and average T-AOC during this period was 4.17 ± 0.99 U mg protein −1 .After exposure to sulphide for 72 h, T-AOC was lower in all treatments compared with the control, especially in the 80 and 160 µM l −1 treatments (p < 0.05).At 96 h exposure, 160 µM l -1 treatments showed higher T-AOC, while 640 µM l −1 treatments showed significantly higher T-AOC than the control (p < 0.05).After releasing the polychaetes from sulphide for 12 h, T-AOC was higher in the 160− 640 µM l −1 treatments relative to the control (p < 0.05).T-AOC of the 640 µM l −1 treatment still remained higher compared with the control (p < 0.05) at 24 h after removal from sulphide.T-AOC showed a similar trend to the control after 48 h.

DISCUSSION
Sulphide is a widely distributed toxicant in aquatic habitats.Sulphide, ammonia, and nitrite are the main contaminations in intensive aquaculture and detrimentally affect cultured organisms and the environment (Bagarinao 1992).Polychaetes have a good ability to resist sulphide stress at higher levels ) Sulphide exposure and removal (h) than those normally found in the natural environment (Miron & Kristensen 1993).The sulphide concentration in the living environment of the polychaete Arenicola marina is 1.7−340 µM l −1 (Völkel & Gries haber 1992).Hauschild & Grieshaber (1997) found that the respiration of A. marina was not significantly affected during exposure to 25 µM l −1 sulphide.Miron & Kristensen (1993) found that the sulphide concentration reached 2000 µM l −1 in the pore water of sediment in the habitat of Nereis succinea and N. diversicolor, but it was no more than 50 µM l −1 in the habitat of N. virens.This result indicates that N. succinea and N. diversicolor can tolerate higher sulphide concentrations than N. virens.Generally, the normal concentration of sulphide in the living environment of polychaetes is fatal for other aquatic animals, such as the crab Eriocheir sinensis, the amphipod Gammarus pseudolimnaeus, and the may - ) 0 µmol l -1 80 µmol l -1 160 µmol l -1 320 µmol l -1 640 µmol l -1 Fig. 5.Total antioxidant capacity (T-AOC) in Perinereis aibuhitensis during and after the period of exposure to different concentrations of sulphide (means ± SD).Abbreviations and definitions are the same as in Fig. 3 fly Hexagenia limbata (Wang & Chapman 1999, Gu 2007), showing that polychaetes can endure higher sulphide concentrations than many other aquatic animals.Moreover, different polychaetes can endure different sulphide concentrations.Our study showed that the tolerance ability of Perinereis aibuhitensis for sulphide is high.The SC level (one-tenth of the LC 50 at 96 h) is considered to be the safe concentration of toxicant that has no detrimental effect on organisms (Sprague 1971).In the present study, we inferred that the tolerance of P. aibuhitensis for potentially toxic H 2 S was 6.91, 5.31, and 0.86 µM l −1 at 15, 20, and 25 °C, respectively.Correspondingly, the safe sulphide concentrations of P. aibuhitensis were 1099.48,454.61, and 67.83 µM l −1 at 15, 20, and 25 °C, respectively.The morphological abnormalities under sulphide toxicity differed from the phenomenon that worms detach their tails when faced with toxic metal contaminants (Tian et al. 2014).It was inferred that death was probably caused by hypoxia in tissues, because biomembranes are highly permeable to sulphide (Beerman 1924), and polychaetes produce significantly more anaerobic metabolites when exposed to sulphide, even at high O 2 tensions (Hauschild & Grieshaber 1997).Moreover, Julian et al. (1998) found that sulphide directly inhibits muscle contraction when studying the neuromuscular sensitivity of Urechis caupo to H 2 S.
In the acute toxicity test, the mortality rate increased with increasing temperature and LC 50 showed a negative relationship with temperature and exposure time.It is known that the permeability of sulphide is high (Vismann 1990).Sulphide detoxification is highly oxygen-dependent (Hauschild & Grieshaber 1997) and is an important way to reduce the conversion of toxic sulphide to thiosulphate (Johns et al. 1997).Since polychaete respiration increases at higher temperatures (Fadhullah & Syakir 2016), this may result in severe hypoxia and higher mortality in polychaetes.The mortality rate of polychaetes exposed to sulphide was not affected by salinity (24−30) in the present study.Activation of antioxidant enzymes (SOD, CAT, T-AOC) in marine organisms is an important mechanism to resist various types of oxidative stress, including sulphide stress (Wang & Chapman 1999, Vidal-Liñán et al. 2010, Qiu et al. 2013).Xu et al. (2014) found that sulphide significantly affects several enzyme activities (SOD, CAT, lysozyme (LSZ), and malondialdehyde (MDA)) of Charybdis japonica, which can be used as indicators of the immune state under sulphide contamination.SOD can catalyze the decrease of reac-tive O 2 • − by transferring it to H 2 O 2 , which is also an important ROS.Furthermore, H 2 O 2 can subsequently be detoxified by CAT.T-AOC is another antioxidant enzyme that can eliminate ROS to prevent lipid peroxidation and decompose peroxide (Xu & Pan 2013).
It has been proposed that sulphide alters antioxidant activities by inhibiting functional en zymes (Sun et al. 2014).Our study showed that SOD activity decreased during 72 h sulphide exposure, but increased at 96 h exposure.After removal from sulphide for 12 h, SOD activity of most treatments remained at a relatively low level.It was interesting that CAT also showed lower activity after 72 h of recovery.Previous studies have shown that the CAT activity of Urechis unicinctus recovers within 24 h after removal from sulphide exposure when it is exposed to 50 and 150 µM l −1 of sulphide for 48 h (Wang 2006).Völkel & Grieshaber (1994) found that the influx of sulphide into the coelomic fluid of A. marina can be oxidized sufficiently under normoxia when sulphide concentrations are lower than 330 µM l −1 .Otherwise, the sulphide concentration would be 5 µM l −1 in the coelomic fluid when external sulphide concentrations exceed that level.In this study, the results showed that a high sulphide concentration and long sulphide exposure required a longer time to eliminate ROS by increasing SOD and CAT activity.Moreover, when polychaetes were exposed to sulphide concentrations below 320 µM l −1 , they were able recover within 24 h after removal from sulphide, otherwise, it took longer.
Following oxidative damage of lipids, proteins, and other molecules, ROS-induced stress can be eliminated by cellular antioxidant enzymes including CAT, SOD, and the glutathione (GSH) system (Duval et al. 2002).In the present study, we found that CAT activity was more sensitive to low concentrations of sulphide in the initial 6 h of exposure; however, no significant changes were observed in the higher concentration treatments (320 and 640 µM l −1 ).A similar result was also found when P. aibuhitensis was exposed to Pb 2+ (Tian et al. 2014).Previous studies have suggested that CAT activity increases when living organisms suffer toxic oxidative stress (Nusetti et al. 2005, Tian et al. 2014).In our study, increased CAT activity was found at 12 h of exposure, which possibly counterbalanced sulphide-induced ROS production.Furthermore, CAT activity decreased while SOD activity increased at 96 h sulphide exposure.Therefore, we inferred that SOD neutralized ROS instead of CAT at this time.Nusetti et al. (2005) also found that the existence of other enzymatic and nonenzymatic antioxidants played a function in stead of GSH activity, which could be influenced by glutathione reductase (GR) activity.CAT activity in all treatments was lower than that in the control during the initial 6 h of the recovery period, which seemed to be an extension of those at 96 h exposure.Then the CAT activity in creased after 12 h of recovery.Moreover, the lower-concentration treatments (80 and 160 µM l −1 ) maintained relatively low CAT activities during the 24−96 h recovery period, while the higherconcentration treatments, especially the 640 µM l −1 treatment, presented lower CAT activities until 48 h recovery.Thereby, we inferred that polychaetes require more time to recover from higher concentrations of sulphide stress.The results were consistent with our observations that the vitality of polychaetes, especially in the higher-concentration treatments (320 and 640 µM l −1 ), decreased after 48 h of exposure to sulphide and increased after 24 h of removal from sulphide.
T-AOC is a comprehensive reflection of the enzymatic and non-enzymatic antioxidant system (Zhou et al. 2014).In our study, T-AOC in P. aibuhitensis showed an increasing trend at 6 h exposure.Then it showed no difference from the control during 12−72 h exposure, and began to increase in the higher-concentration treatments at 96 h sulphide exposure.T-AOC was at a higher level than the control at 12 h removal from the sulphide.Thus, we inferred that T-AOC eliminates ROS at the beginning of exposure to sulphide and returns to a steady level after 24 h of recovery.Sies (2007) proposed that the antioxidant enzyme system maintains a steady state of metabolites and functional integrity, which is organized into 3 principal levels of protection: prevention, interception, and repair.In our study, vitality of polychaetes was reduced as exposure time and sulphide concentration increased, which was more obvious after 48 h exposure in the higher sulphide concentration treatments (>320 mmol l −1 ).Considering en zyme activity and vitality of polychaetes, enzyme activities are possibly responsible for the restoration of oxidative balance, because vitality is restored after 24 h of recovery.It is clear that P. aibuhitensis has a strong selfdetoxification system to cope with sulphide toxicity.
The increasing demand for remediation of sulphide contamination caused by intensive coastal aquaculture has led to increased interest in the ability of burrowing polychaetes to increase oxygen in sediment and thus reduce sulphide (Grieshaber & Völkel 1998).Sulphide concentration in the habitat of P. aibu hitensis may be lower than the concentrations found in the present study.S 2− can combine with metal ions, such as Pb 2+ , Cu 2+ , Cd 2+ , and Zn 2+ in the form of metal− sulphide complexes that are more oxidized and more labile.Metal− sulphide complexes are considerably less bioavailable than metal ion contaminants; thus, these meta-sulphide complexes are more toxic (Edwards et al. 2013, Simpson & Spadaro 2016).Moreover, Joyner-Matos et al. (2010) found when worms Glycera dibranchiata were exposed in vivo to 0−10 mmol l −1 sulphide for 24 h, oxidative damage to RNA and DNA in the body wall tissue occurred and coelomocytes were in creased, which showed sulphide could be an environmental mutagen.Further research is needed to better understand how polychaetes detoxify sulphide, including ecophysiological studies on sulphide detoxification and metabolic pathways of polychaete in sulphidic habitat environments.

Fig. 4 .
Fig.4.Catalase (CAT) activity in Perinereis aibuhitensis during and after the period of exposure to different concentrations of sulphide (means ± SD).Abbreviations and definitions are the same as in Fig.3

Table 2 )
, while the LC 50 value of H 2 S decreased significantly with increasing temperature

Table 1 .
LC 50 and safe concentration (SC) of H 2 S to Perinereis aibuhitensis and their 95% confidence limits under different water temperatures and salinities.LC 50 : concentration that causes mortality in 50% of organisms.The SC was calculated based on the following formula: SC = 96 × LC 50 × 0.1

Table 2 .
Two-way ANOVA (main effect) results of 24 h LC 50 , 48 h LC 50 , 72 h LC 50 , and 96 h LC 50 of H 2 S at different temperatures and salinities (Salt) Fig.3.Superoxide dismutase (SOD) activity in Perinereis aibuhitensis during and after the period of exposure to different concentrations of sulphide (means ± SD). S = exposure to sulphide; R = removal from sulphide.The numbers after S and R are hours of exposure/removal.Different letters denote significant differences among different sulphide concentrations at the same exposure or recovery times (p < 0.05)