Tetramethylthiuram monosulphide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Additional information

The chronic toxicity to fish of tetramethylthiuram monosulphide was assessed by Van Leeuwen et al. (1986, 1990). However, these studies were disregarded for many reasons (significant methodology deficiences and documentation insufficient for assessment).

As a conclusion of the critical reviewing of Van Leeuwen et al. (1986, 1990), we consider that both studies should be both classified Klimisch 3a (documentation insufficient for assessment) and 3b (significant methodological deficiencies). They should therefore be disregarded for regulatory purposes under REACH regulation (see below our position paper ; it is also attached as a pdf file in this section).

POSITION PAPER ON TWO ACADEMIC RESEARCH LITERATURE ARTICLES ON THE TOXICITY OF TETRAMETHYLTHIURAM FOR FISH EARLY LIFE STAGES.

This position paper deals with the following articles:

 - Van Leeuwen C.J. et al., Aquatic toxicological aspects of dithiocarbamates and related compounds. III. Embryolarval studies with rainbow trout (Salmo gaidneri); Aquatic Toxicology 9, 129-145 (1986).

- Van Leeuwen C.J. et al.,Fish Embryos as Teratogenicity Screens: A comparison of Embryotoxicity between Fish and Bird; Ecotoxicology and Environmental Safety 20, 42-52 (1990).

These studies investigated the effects of tetramethylthiuram monosulphide on the embryo and larval developments of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). LOEC were determined for some regulatory endpoints. However, the articles are not reliable enough to consider their results as relevant for regulatory purposes. We detail below the reasons why we conclude for disregarding these studies and classify them as Klimisch 3a/3b.

I – Critical analysis of van Leeuwen et al. (1986) Aquatic toxicological aspects of dithiocarbamates and related compounds. III. Embryolarval studies with rainbow trout (Salmo gaidneri). Aquatic Toxicology 9, 129-145.

Study description

Van Leeuwen et al. (1986) carried out a 60-days ELS test on rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) eggs and larvae under semi-static conditions, similar to the OECD 210 (2013) fish early-life toxicity test guideline. Freshly spawned eggs were challenged with graded concentrations of tetramethylthiuram monosulphide dissolved in dimethylsulfoxide (DMSO) and separated by a 1.8 spacing factor. Eggs were incubated in relative darkness at 10°C and larvae hatched after 32 days. Photoperiod was then set at 12L:12D and kept constant until the end of the experiment. “Embryotoxicity”, mortality, weight and length of surviving larvae at the end of the experiment were recorded. This study is quoted Klimisch 3 for a multiplicity of reasons that are detailed below.

Inappropriate energy status of fish

Guideline 210 requires to feed rainbow trout larvae from the swim-up stage, a step that usually occurs 10 to 14 d after hatching (2015 FAO), i.e. at days 42 to 46 of the study, which was not done in the study of van Leeuwen et al. (1986).The absence of feeding for the 14 to 18 remaining days certainly resulted in lower energy status and weakened organisms, which is in opposition with the principle of the test.

Photoperiod was set at 12L:12D just after hatching, while a 7-day darkness period is demanded in the OECD 210 guideline. As for feeding, the 7-day darkness period is set to limit the energy loss due to the movements of the larvae under light conditions. The larvae are indeed more active under light than under darkness, which induces supplemental energy consumption and faster yolk sac depletion.

These two biological limitations surely affected the results by increasing the sensitivity of fish to TMTM in an inappropriate way for regulatory purposes.

Methodological deficiencies

The authors used DMSO as a solvent to dissolve TMTM. According to most OECD and ECHA’s guidances, the use of a solvent should be avoided in aquatic toxicity testing. For instance, OECD Guidance document n°23 on aquatic toxicity testing of difficult substances and mixtures indicates that “because of the potential for interaction with the test substance resulting in an altered response in the test, their use should be restricted to situations where no other acceptable method of media preparation is available”. TMTM solubility is 308 mg/L, which is more than enough to avoid the use of any organic solvent for aquatic toxicity testing. Moreover, Kais et al. (2013) recently showed that low concentrations of DMSO down to 0.1% can facilitate the entrance of exogenous substances through the chorion of zebrafish eggs. They however concluded that the use of a maximum 0.01% concentration DMSO, as recommended for regulatory purposes of the fish embryo toxicity test (FET) is safe with zebrafish. But the investigated incubation time had only a 2-day duration. In rainbow trout, incubation time is 16 times longer (32 days). It could therefore be possible that the down limit of safe use of DMSO in rainbow trout ELS test is lower than 0.01% which is the DMSO concentration used in the study of Van Leeuwen et al. (1986). Kais et al. (2013) themselves concluded that their study shades doubts about the impact of DMSO in aquatic toxicity testing with fish eggs requiring long-time incubation periods. The use of DMSO by Van Leeuwen et al. (1986) is therefore considered as a methodological deficiency for regulatory purpose because (i) its use was not necessary with regards to TMTM solubility and (ii) it may have weakened embryo’s resistance, which is contrary to the principle of aquatic toxicity testing for regulatory purposes under REACH regulation.

Dissolved oxygen was not monitored throughout the study. This clearly is a major experimental flaw when working with fish in general. A hundred rainbow trout larvae kept in 10 L water is indeed a significant loading rate requiring care to water oxygen level. Even if the authors state that aeration was provided, this is not sufficient proof that adequate oxygen concentrations were maintained throughout the 60 days of the study. This lack of information is considered as a methodological deficiency.

Loading rate was a hundred larvae per 10 L of solution in a semi-static system. Solution renewal was every two or three days. Assuming a larval mean weight of 50 mg at hatching as a lower case (From and Rasmussen, 1991), this makes a loading rate of 0.5 g/L over a 72-h period, i.e. a loading rate of 1.5 g/L per 24-h period, which is far above the recommended 0.5 g/L per 24 hours (OECD 210, 2013). This raises concerns about water quality (not monitored) and also dissolved oxygen levels, as discussed above.

Other methodological deficiencies include (i) duplicate instead of the quadruplicate testing recommended by the OECD 210 (2013) guideline, (ii) absence of water temperature monitoring and (iii) absence of any analytical monitoring of the substances.

Documentation insufficient for assessment

The article only presents statistical conclusions. No figure, table or data about the analysed variables (mortality, embryotoxicity, larval weight, larval length) in function of the tested doses are presented. Therefore, no judgement about the severity of the dose effect can be made (e.g., do the significant effects detected by mean comparison correspond to 3, 20% or any other percentage difference with the control group?).

The range of tested concentrations is not given and cannot be accurately guessed (although we would agree using a LOEC if the study and article were of sufficient quality for regulatory purposes). The only information given is the spacing factor which is 1.8

The quality (hatching and survival rates) of both control groups (with and without solvent) cannot be assessed. The authors recognized that they had some mortality in the control groups without giving any figure.

The monotonicity of the dose-response and statistical regression quality cannot be checked.

It is not known if the solvent control is of adequate quality (hatching and survival rates).

The control used for mean comparison is not mentioned (solvent-free control, solvent control or both ?).

The LOEC for length and weight are different, which is abnormal since both parameters are normally highly correlated in fish.

We consider that the documentation provided by van Leeuwen et al. (1986) is insufficient for reliability assessment.

Conclusions

For all these reasons, this study is classified Klimisch 3a (documentation insufficient for assessment) and 3b (significant methodological deficiencies). It is therefore disregarded for regulatory purposes in the framework of the REACH regulation. We consider that the results of Van Leeuwen et al. (1986) should not be taken into account for PNEC derivation.

II – Critical analysis of van Leeuwen C.J. et al. (1990)Fish Embryos as Teratogenicity Screens: A comparison of Embryotoxicity between Fish and Bird; Ecotoxicology and Environmental Safety 20 ,42-52.

Study description

Van Leeuwen et al. (1990) carried out a 60-days ELS test on rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) eggs and larvae and a 7-days ELS test on zebrafish Danio rerio. The study on rainbow trout is in fact the same as described in van Leeuwen et al. (1986) (see commentary in the first section). The study on zebrafish is not a real long-term toxicity to fish experiment but we still examined the protocol description and results. Freshly spawned eggs of zebrafish were challenged with different doses of tetramethylthiuram monosulphide dissolved in DMSO and separated by a 3.2 spacing factor. Sixty eggs were incubated in 50 mL solution for each concentration. Photoperiod was then set at 12L:12D and kept constant until the end of the experiment. Temperature was set at 25°C.Treatments were not replicated. “Embryotoxicity” and larval mortality were recorded.

We consider that this study should be quoted Klimisch 3 for a multiplicity of reasons that are detailed below.

Methodological deficiencies

The authors used DMSO as a solvent to dissolve TMTM. According to most OECD and ECHA’s guidances, the use of a solvent should be avoided in aquatic toxicity testing. For instance, OECD Guidance document n°23 on aquatic toxicity testing of difficult substances and mixtures indicates that “because of the potential for interaction with the test substance resulting in an altered response in the test, their use should be restricted to situations where no other acceptable method of media preparation is available”. TMTM solubility is 308 mg/L, which is more than enough to avoid the use of any organic solvent for aquatic toxicity testing. Moreover, Kais et al. (2013) recently showed that low concentrations of DMSO down to 0.1% can facilitate the entrance of exogenous substances through the chorion of zebrafish eggs. They however concluded that the use of a maximum 0.01% concentration DMSO, as recommended for regulatory purposes, is safe with zebrafish. But the investigated incubation time had only a 2-day duration. In van Leeuwen et al. (1990), incubation time until hatching was twice longer (4 days). It could therefore be possible that the bottom limit of safe use of DMSO in this study is lower or close to 0.01% which is the DMSO concentration used in the study of Van Leeuwen et al. (1990). The use of DMSO by Van Leeuwen et al. (1986) is considered as a methodological deficiency for regulatory purpose because (i) it was not necessary with regards to TMTM solubility and (ii) it may have weakened embryo’s resistance, which is contrary to the principle of aquatic toxicity testing for regulatory purposes under REACH regulation.Dissolved oxygen was not monitored throughout the study. This clearly is a major experimental flaw when working with fish in general. Sixty zebrafish larvae confined in 50 mL solution is indeed a significant loading rate requiring care to water oxygen level. Even if the authors state that aeration was provided, this is not sufficient proof that adequate oxygen concentrations were maintained throughout the 7-day period of the study. This is considered as a methodological deficiency.

Treatments were not replicated which is a major flaw. We wonder how mean comparisons could be performed without any replicate (e.g. how can a LOEC for mortality rate be calculated without any tank replicate ?). This is probably one of the highest concerns that can be raised about this study.

Other methodological deficiencies include (i) absence of water temperature monitoring and (ii) absence of any analytical monitoring of the substances.

Documentation insufficient for assessment

In this section, we address the same critical remarks to van Leeuwen et al. (1990) as to van Leeuwen et al. (1986) (see part I of the critical review). We consider that the documentation provided by van Leeuwen et al. (1990) is insufficient for reliability assessment.

Conclusions

For all these reasons, this study is classified Klimisch 3a (documentation insufficient for assessment) and 3b (significant methodological deficiencies). It is therefore disregarded for regulatory purposes under REACH regulation. We consider that the results of Van Leeuwen et al. (1990) should not be taken into account for PNEC derivation.

III - Overall conclusions

As a conclusion of the critical reviewing of Van Leeuwen et al. (1986, 1990), we consider that both studies should be both classified Klimisch 3a (documentation insufficient for assessment) and 3b (significant methodological deficiencies). They should therefore be disregarded for regulatory purposes under REACH regulation.

References:

FAO, 2015:http://www.fao.org/fishery/culturedspecies/Oncorhynchus_mykiss/en

From J., Rasmussen G., 1991. Growth of rainbow trout,Oncorhynchus mykiss(Walbaum, 1792) related to egg size and temperature. Dana 9, 31-38.

Kais B., Schneider K.E., Keiter S., Henn K., Ackermann C., Braunbeck T., 2013. DMSO modifies the permeability of the zebrafish (Danio rerio) chorion – implications for the fish embryo test (FET). Aquatic Toxicology 140-141, 229-238.

OECD, 2000.Guidance document on aquatic toxicity testing of difficult substances and mixtures.Series Testing and Assessment, No. 23, Organisation for Economic Co-operation and Development, OECD,.

OECD 210, 2013.Fish, Early-Life Stage Toxicity Test. Organisation for Economic Co-operation and Development, OECD,.