Tetrakis(phenylmethyl)thioperoxydi(carbothioamide)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

-Molecular formula: C30H28N2S4
-Molecular weight: 544.821
-CAS Number: 10591-85-2
-Description: Off-white powder
-Test substance storage: At room temperature in the dark
-Stability under storage conditions: Stable
-Batch: 80600108
-Purity: 97.7%
-Expiry date: 01 June 2010

Analytical monitoring:

yes

Details on sampling:

One day before the start of the test a sample was taken from the test medium to check the functioning of the flow-through system. During the reproduction test samples for analysis were taken from the test medium and the treatment-control. The method of analysis is described in the appended Analytical Report (Appendix V).

Sampling:
- Frequency: at the start, after 7, 14 and 21 days of exposure.
- Volume: 500 ml
- Storage: Not applicable, samples were analysed on the day of sampling.

Vehicle:

yes

Details on test solutions:

Stability in vehicle:
• Water: Unknown
• Acetone: At least 96 hours

Solubility in vehicle:
• Water: Very low, 0.001-0.01 mg/l (20.5°C)
• Acetone: Yes

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. precipitate or a film of the test substance on the water surface).

The batch of TBzTD tested was an off-white powder with a purity of 97.7%, which was tested at or close to its limit of solubility in water. Stock solutions of TBzTD in Acetone (Merck, Darmstadt, Germany) were weekly prepared at a concentration of 0.1 mg/ml. No other treatment than careful shaking was necessary to completely dissolve the stock in Acetone resulting in a clear and colourless solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- Strain/clone:
- Source: In-house laboratory culture with a known history.
- Food type: a suspension of fresh water algae (Chlorella pyrenoidosa)
- Frequency: daily - 0.4 mgC/Daphnia/day after correction for the wast of algae = twice the recommended value for feeding per daphnia in OECD211)
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Medium: M7 (as prescribed by Dr. Elendt-Schneider)

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

varied between 143 and 268 mg calcium carbonate per litre

Test temperature:

The temperatures in the test media varied between 20.2 and 21.9°C. The temperature continuously measured in the blank-control varied between 20.3 and 23.9°C during the test. Hence, the maximum temperature exceeded the range as laid down in the protocol (18-22°C, constant within 2°C). On average the temperature was 21.8°C, which was rather high, but within the recommended range for testing with daphnia. The test periods with temperature at or slightly above 22°C, including three short periods with temperature above 23°C (between days 13 and 15 with a peak at 23.9°C) were expected not to have affected the well being of the organisms as was confirmed by the normal reproduction numbers and absence of significant mortality in the essential test groups being the treatment-control and the TBzTD test group.

pH:

remained within the range of 7.4 to 8.5 throughout the test

Dissolved oxygen:

remained within the range of 6.1 to 9.4 mg/l during the exposure period

Salinity:

na

Nominal and measured concentrations:

Analyses of the sample taken from the TBzTD concentration one day before the start of the study showed a measured concentration of 6.6 µg/l. This was within the expected range of 1-10 µg/l that was indicated by the sponsor as being the maximum solubility in water.

Analysis of samples taken during the 21-day test period showed measured concentrations that generally varied between 5.5 and 9.0 µg/l. A slightly lower value of 2.6 µg/l was measured in one of two samples taken on day 14 of the study. Somewhat higher values of 18 and 19 µg/l were measured in duplicate samples at the study end. The mean measured concentration was calculated to correspond to 9 µg/l. Based on these results it can be safely concluded that the maximum soluble concentration in test medium was tested.

Details on test conditions:

The total dissolved organic carbon content of blank M7 medium was not determined. TOC was measured in a sample taken from the blank-control which included algae. As a consequence the TOC was 3.8 mg/l, which was expected to derive mainly from the organic carbon of the algae. Therefore, the TOC of blank-medium was expected to be <2 mg/l which complies with the requirement as laid down in the (ISO) guideline (TOC < 2 mg/l).

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (parental mortality)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (parental mortality)

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: (parental body length)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

9 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: (parental body length)

Details on results:

- Mortality of parent animals: 1 out of 20 of the parental daphnids died in the treatment
- No. of offspring produced per day per female:
- Time to first brood release or time to hatch: in controls, on day 5 (= first eggs in brood pouch) and on day 7 (= first brood appeared) (similar responses were seen in the TBzTD treatment group)

Reported statistics and error estimates:

The values for reproduction observed in the various test groups were expressed as mean number of living young per parent. The mean values for reproduction obtained for the test medium were compared to those recorded in the treatment-control on the various days of recording. Further, the length of the parental daphnids (day 21) exposed to the test substance were compared to the treatment-control.

Statistical analysis: Total reproduction on day 21 was tested for normality and for homogeneity of variance and statistically tested using an ANOVA test followed by a mean comparison test (2 Sample T-test). The software used was TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

LC- and EC-values and NOEC: The overall threshold level of effect and the overall NOEC were determined on these results and statistics.

Mean number of young per parent

Day	8	9	10	11	12	13	14	15	16	17	18	19	20	21
blank-control	4.8	4.8	4.8	15.6	17.3	19.9	25.7	26.2	26.2	26.2	41.4	43.1	53.3	55.1
treatment- control	7	7	7	19.7	23.8	39.7	42.2	43.5	43.5	43.5	66.3	83.5	89.5	95.0
treatment	6.9	6.9	6.9	22.5	28.8	45.9	48.3	54.4	54.4	54.4	76.4	89.1	90.3	101.2

The reproduction curve recorded for the TBzTD treatment closely followed the curve of the treatment-control. The curve recorded for the blank-control clearly stayed behind that of the treatment-control and the TBzTD treatment. It was not completely clear why the parental daphnids exposed to the blank-control reproduced less than the parents in the other two groups. A possible reason might be the absence of acetone in the blank-control. Acetone might have had a positive effect on the feeding efficiency, i.e. kept algae better or longer in suspension, resulting in an improved uptake of algae by the parental daphnia. All results obtained in the TBzTD treatment group were related to the treatment (acetone)-control. Note that the validity criterion for total reproduction was met in the treatment-control. Not meeting this criterion for the blank-control does not affect the validity of the study as test results should always be compared to a control treated in the same way as the test group.

Average body lengths (mm) of parental daphnids with the standard deviation (SD) per concentration measured at the end of the test and the percentage reduction of body length relative to the treatment-control.

Test group	Average length (mm)	SD	% reduction
blank-control	3.55	0.18	na
treatment-control	3.83	0.13	-
treatment	3.77	0.17	2

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present flow-through study, TBzTD did not affect reproduction or survival of Daphnia magna at a mean measured concentration of 9 µg/l after 21 days of exposure.
According to information supplied by the sponsor the maximum solubility of TBzTD in water is between 1 and 10 µg/l. It can therefore be concluded that the mean measured concentration of 9 µg/l corresponds to the maximum solubility of TBzTD and that the NOEC is ≥ 9 µg/l.

Executive summary:

Daphnia magna, 21-day reproduction study with TBzTD.

The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 1998. In addition, the procedures were designed to meet the test methods and validity criteria of the ISO International Standard 10706, 2000, the Council Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The batch of TBzTD tested was an off-white powder with a purity of 97.7%, which was tested at or close to its limit of solubility in water applying a flow-through test system to maintain the concentration at this level.

In the flow-through system the dilution water (M7 medium) was dosed separately from the test substance stock solution into mixing vessels. Weekly a TBzTD stock solution in Acetone was prepared at 0.1 mg/ml. The dosed volumes and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily.

The experimental set-up included stainless steel vessels with 4 stainless steel mesh containers per test group. Each container contained five neonate (< 24h old) Daphnia magna. The target test concentration of TBzTD was 10 µg/l. A blank-control and a treatment-control (Acetone) were also included. The study duration was 21 days and the test solutions were renewed at a flow rate of 2 litres/hour. The daphnids were fed continuously with Chlorella pyrenoidosa (~0.4 mg organic C per daphnid per day). Every workday the condition of the parental daphnids was recorded and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

Analysis of samples taken weekly during the 21-day test period showed measured concentrations that generally varied between 5.5 and 9.0 µg/l. A slightly lower value of 2.6 µg/l was measured in one of two samples taken on day 14 of the study. Somewhat higher values of 18 and 19 µg/l were measured in duplicate samples at the study end. The mean measured concentration was calculated to correspond to 9 µg/l. Based on these results it can be safely concluded that the maximum soluble concentration in test medium was tested.

No significant mortality occurred in any of the three groups tested (mortality ≤ 5%). The average cumulative number of young per female in the treatment-control after 21 days was 95 ± 12. Mean reproduction numbers in the TBzTD treatment group were slightly higher at 101 ± 19. The reproduction between these two groups was not statistically different. Mean parental body length of the parental daphnia obtained in the controls and the TBzTD test group did not differ. There were some recordings of immobile young in each of the three groups tested. However, numbers were relatively low and not considered significant. Unhatched (aborted) eggs were not observed in any of the test groups throughout the 21-day test period.

The study met the acceptability criteria prescribed by the protocol except for two minor deviation which had obviously no impact on the valiadity. Therefore, the study is considered valid.

Under the conditions of the present flow-through study, TBzTD did not affect reproduction or survival of Daphnia magna at a mean measured concentration of 9 µg/l after 21 days of exposure. According to information supplied by the sponsor the maximum solubility of TBzTD in water is between 1 and 10 µg/l. It can therefore be concluded that the mean measured concentration of 9 µg/l corresponds to the maximum solubility of TBzTD and that the NOEC is ≥ 9 µg/l.

Description of key information

Under the conditions of the present flow-through study, TBzTD did not affect reproduction or survival of Daphnia magna at a mean measured concentration of 9 µg/l after 21 days of exposure. 
According to information supplied by the sponsor the maximum solubility of TBzTD in water is between 1 and 10 µg/l. It can therefore be concluded that the mean measured concentration of 9 µg/l corresponds to the maximum solubility of TBzTD and that the NOEC is ≥ 9 µg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9 µg/L

Additional information

Under the conditions of the present flow-through study, TBzTD did not affect reproduction or survival of Daphnia magna at a mean measured concentration of 9 µg/l after 21 days of exposure.

According to information supplied by the sponsor the maximum solubility of TBzTD in water is between 1 and 10 µg/l. It can therefore be concluded that the mean measured concentration of 9 µg/l corresponds to the maximum solubility of TBzTD and that the NOEC is ≥ 9 µg/l.