Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2018 - ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: Clear, yellow liquid
Purity: >99% (UVCB)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and 100 mg/L loading rate WAF group from fresh and old media throughout the 96 hour test period for quantitative analysis. The samples were stored frozen prior to analysis. Duplicate samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST MEDIUM
Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened, giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

The test water used for both the range finding and definitive tests was the same as that used to maintain the stock fish.

PREPARATION AND APPLICATION OF TEST SOLUTION
Due to the low aqueous solubility and the complex nature of the test item, the test medium was prepared as Water Accommodating Fraction ((WAF) based on a the methods established in a preliminary mixing trial.

A nominal amount of test item was added to the surface of test water to achieve a nominal loading rate of 100 mg/L. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Visual observations of the WAF indicated that a significant amount of dispersed test item was present in the water column and hence, it was considered justifiable to remove the WAF by filtering through a glass wool plug and a sheet of filter paper. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded), then filtering twice through filter paper to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering which showed that no undissolved material remained in the water column, with the exception of the solution preparation for the first 24-hour renewal interval where the media was observed to be a very slightly hazy dispersion, however, it was considered that further filtration would be unsuccessful at removing any further dispersed material. During the second, third and fourth preparations, filter paper was used to remove all undissolved material, with microscoptic observations carried out after filtration showing that no undissolved material remained.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST SYSTEM AND SUPPORTING INFORMATION

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 April 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 07 May 2018 to 15 May 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. The water temperature was controlled at approximately 14 °C to 15 °C with a dissolved oxygen content of greater than or equal to 9.7 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 19 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.1 cm (standard deviation = 0.2) and a mean weight of 1.05 g (standard deviation = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.37 g body weight/liter.

The diet and test water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

140 mg/L as CaCO3

Test temperature:

14 °C to 15 °C

pH:

7.3 - 8.1

Dissolved oxygen:

9.9 - 10.6 mg O2/L

Nominal and measured concentrations:

Nominal loading rates (WAF) were 0 (control) and 100 mg/L

Analysis of the freshly prepared test preparations at 0 and 72 hours (see Annex 4) showed that measured concentrations of 1.1 and 0.054 mg/L were obtained, respectively, and of the old or expired media at 24 and 96 hours showed that measured concentrations of 0.85 and 0.026 mg/L were obtained, respectively.

A higher measured concentration was obtained in the 0-24 hour test preparations. Inspection of the data showed that despite the media being filtered through a glass wool plug and twice through filter paper, undissolved material remained in the water column. This would account for the higher measured concentration observed in the first test solution renewal interval. Given that the presence of the undissolved material did not result in any toxicity, this was considered not to have had an impact on the outcome or integrity of the test.

The dissolved test item may have been comprised of one or several components of the test item, two of which could be measured by LC-MS. Given that toxicity cannot be attributed to a single component or specific mixture of components but to the test item as a whole, the results were based on nominal loading rates.

Details on test conditions:

An initial range-finding test was conducted, however, daily renewal of the test preparations was not performed in error and as such, the test was terminated prior to completion. The results of this initial range-finding test were not used for reporting purposes.

Based on the results of a second preliminary range finding test, in which groups of three fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L and no toxicity was observed the definitive test was conducted as a "Limit test" at a single loading rate of 100 mg/L to assess fish toxicity under the same exposure conditions.

Glass exposure vessels (25 - 30 liters) containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test solutions to aid in maintaining exposure levels and prevent the build-up of nitrogenous waste products.

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Details on results:

Exposure of rainbow trout to the test item for 96 hours resulted in an LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary to test at loading rates in excess of 100 mg/L.

There were no sub-lethal effects of exposure observed in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of rainbow trout to the test item for 96 hours resulted in an LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary to test at loading rates in excess of 100 mg/L.

Executive summary:

A study was performed to the standard OECD Guideline 203 and EU Test Method C.1, under GLP

conditions to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss).

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the

test solutions were prepared as a Water Accommodated Fractions (WAF).

Following a preliminary range‑finding test, seven fish were exposed to a WAF of the test item at a single nominal loading rate of100 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi‑static test conditions. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours

The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard; quantitation was based on the peak of the major constituent detected. Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of 1.1 and 0.054 mg/L were obtained, respectively, and of the old or expired test preparation

at 24 and 96 hours showed that measured concentrations of 0.85 and 0.026 mg/L were obtained, respectively.

The dissolved test item may have been comprised of one or several components of the test item. Given that toxicity cannot be attributed to a single component or a specific mixture of components, the results were

based on nominal loading rates only.

Exposure of rainbow trout to the test item for 96 hours resulted in an LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

A study was conducted according to OECD Guideline 203 and EC Test Method C.1, under GLP, to assess the acute toxicity of the test item to the freshwater fish, rainbow trout (Oncorhynchus mykiss) using the limit test approach. The 96 hour LL50 value was determined to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) rate was 100 mg/L loading rateWAF (Envigo, 2018).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L