2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Preferred study for this SIDS endpoint; guideline study conducted under GLP. The 50% and 100% WAF solutions were analyzed on the basis of total tin and were converted to and reported as the test substance, DMT(2-EHMA). The preparation of the WAF allowed both the soluble portion of the test substance and the hydrolysis products to be present in the medium for the chronic study. The 3 lowest test concentrations, 1% ,5%, and 10% WAF, were below the analytical limit of detection (LOD) (2.3 ug/L); a value of one-half the LOD was used to calculate the Sn concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The deviations did not adversely affect the study

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 5, 10, 50, and 100% of a WAF prepared at a loading rate of 100 mg/L. Controls – test medium without test substance or other additives (0 mg/L).
- Sampling method: During the study duplicate samples were taken from all test concentrations and the blank-control at the beginning and end of three intervals of 48 hours (nominal days 0 and 2, 5 and 7, 12 and 14) and of an interval of 72 hours (nominal days 16 and 19).
- Sample storage conditions before analysis: Samples were stored at room temperature until transportation to TNO Nutrition and Food Research.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Fresh test solutions were prepared three times a week, starting with a stock at a loading rate of 100 mg/L. This stock solution was magnetically stirred for 24 hours, after which it was left to stabilize for another 24 hours. The water fraction was then separated from the undissolved fraction of test substance using a pipette force. The water fraction was filtered over a glass filter (GF/C Whatman). The filtrate was diluted in M7 medium to reach the lower test concentrations.
MONITORING OF TEST SUBSTANCE CONCENTRATION: Duplicate samples were taken from all test concentrations and the control at the beginning and end of three intervals of 48 h (nominal days 0 and 2, 5 and 7, 12 and 14) and of an interval of 72 h (nominal days 16 and 19). Undiluted WAF additionally at the start of the 2nd, 4th, 5th, 7th, and 9th interval (nominal days 2, 7, 9, 14, and 19).
- Differential loading: 100 mg/L
- Controls: Test medium without test substance or other additives (0 mg/L)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): NA
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1, 5, 10, 50, or 100% of the WAF prepared at 100 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The batch of DMT (EHMA) tested was a white hazy liquid with a purity of 87.0% and not completely soluble in test medium at the concentrations tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna, (Crustacea, Cladocera)
- Justification for species other than prescribed by test guideline: This system has been selected as an internationally accepted species.
- Source: Culture of the performing laboratory
- Age of parental stock (mean and range, SD): Breeding- start of each batch- With newborn daphnids, i.e. less than 3 days old, by placing them individually into 50 mL of medium in an all-glass culture vessel.
- Feeding during test
- Food type: A suspension of fresh water algae (Chlorella pyrenoidosa)
- Amount: 0.22-0.24 mg C/daphnid/day
- Frequency: Daily


ACCLIMATION
- Acclimation period: No data
- Acclimation conditions (same as test or not): No data
- Type and amount of food: A suspension of fresh water algae (Chlorella pyrenoidosa)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No data

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- Start of each batch- with newborn daphnids, i.e. less than 3 days old, by placing them individually into 50 mL of medium in an all-glass culture vessel.
- Maximum age of the cultures - 4 weeks.
- Renewal of the cultures- After 7 days of cultivation half of the medium twice a week.
- Temperature of medium - 18-22◦C, constant within ± 1◦C
- Feeding - Daily, a suspension of fresh water algae (Chlorella pyrenoidosa)
- Validity of batch – Frequent inspection with respect to number of young, and appearance of young and parental daphnids.
- Medium – M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

not applicable

Hardness:

232-286 mg CaCO3/L

Test temperature:

19.2-20.2°C (test media) and 18.2-20.3°C (room temperature)

pH:

7.5-8.1

Dissolved oxygen:

>6.0 mg/L

Salinity:

no data

Nominal and measured concentrations:

0, 1, 5, 10, 50, and 100% WAF with a loading rate of 100 mg/L
0, 49, 231, 457, 2316, and 4879 micrograms DMT(EHMA)/L (calculated)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: All glass – no other information reported
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): Three times per week over the 21 days of testing
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): duplicate
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): duplicate
- Biomass loading rate: Fresh test solutions were prepared three times a week, starting with a stock at a loading rate of 100 mg/L.


TEST MEDIUM
M7 TEST MEDIUM
- Source: Prepared in the laboratory according to Elendt (1990).
- Aeration: Not reported.
- Alkalinity: Not reported.
- Hardness: 250 mg CaCO3/L.
- TOC: <2 mg/L.
- Ca/Mg ratio: Not reported.
- Na/K ratio: Not reported.
- TSS: Not reported.
- pH: 8.0 ± 0.2
- Oxygen content: Not reported.
- Conductance: Not reported.
- Holding water: Not applicable.


OTHER TEST CONDITIONS
- Adjustment of pH: The pH remained within a range of 7.5 to 8.1 throughout the study and thus was maintained within the limits prescribed by the protocol (6.0-9.0, constant within 1.5 units).
- Photoperiod: 16 hour photoperiod daily
- Light intensity: 530 and 780 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
OBSERVATIONS: PARENTAL
- Mortality: Every working day the dead as well as the living daphnids were recorded.
- Presence of eggs in the brood pouch: Every working day.
- Body length: At the end of the test.
OBSERVATIONS: F1 GENERATION
- Appearance of first brood: When observed.
- Newborns: Every working day the number of newborn daphnids was counted and their condition was recorded.
- Presence of unhatched eggs: When observed.
- Mortality: When observed.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2 316 µg/L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

457 µg/L

Basis for effect:

other: reproductive output, mortality, body length

Details on results:

EXPOSED In the three lowest treatment groups, the test concentrations were sometimes below the limit of detection (LOD = 4.5 ug/L); half the LOD (2.3 ug/L) was used to calculate the Sn concentration.

- Mortality (parental): 20% (1% WAF), 30% (5% WAF), 10% (10% WAF), 100% (50% WAF), and 100% (100% WAF).
Three out of the twenty parental daphnids died during the test period in the control. Treatment-related mortality occurred at 2316 and 4879 µg DMT (EHMA)/L, while mortality at lower concentrations was ≤ 30%. The cumulative number of deaths is given below:
Concentration DMT(EHTG) Exposure (ug/L) Number exposed Mortality %
0 mg/L (Blank) 0 20 15
1% 49 10 20
5% 231 10 30
10% 457 10 10
50% 2316 10 100
100% 4879 10 100
- Appearance of first brood: day 8.
- Reproduction: There was no significant reduction in the mean cumulative number of young per parent at the three lowest test substance concentrations. At the two highest test concentrations, reproduction could not be recorded due to total mortality. The first young in the control were observed on day 8. The reproduction curves recorded at the three lowest treatment levels closely followed the curve of the control, and the reproduction was not inhibited, but comparable or even higher than in the control.
- Body length: No data is available for the two highest treatments groups, as all daphnids in these groups died during the test. In the other treatments, the mean body length was not significantly reduced relative to the control.
Concentration DMT(EHTG) Exposure (ug/L) Average length (mm) SD % Reduction
0 mg/L (Blank) 0 4.27 0.14 ---
1% 49 4.29 0.16 -0.5
5% 231 4.35 0.12 -1.9
10% 457 4.28 0.17 -0.4
50% 2316 n.a. n.a. n.a.
100% 4879 n.a. n.a. n.a.
- Small sized: None, however, a dose-dependency of parental condition was observed (daphnids were pale and trapped at the surface).
- Unhatched eggs: At 5% WAF, one daphnid produced unhatched eggs.
- Mortality (offspring): At 5 and 10% WAF, one immobile neonate was produced on day 19.
- Number of males and females (parental): on Day 21: 18, 8, 7, 9, 0 and 0 for the ocntrol, 1%, 5%, 10%, 50% and 100% of the WAF, respectively.
- Effect concentrations exceeding solubility of substance in test medium: Test solutions were not noted to contain a floating layer. It should be noted that no undissolved test substance or floating layer was observed in the test media.
Other Parameters – Aborted eggs were observed in one control vessel on day 19, and in one vessel at 231µg DMT(EHMA)/L on day 20. No further unhatched eggs or immobile young were observed at any of the concentrations tested.
In the control and at the lowest treatment level (49 µg DMT(EHMA)/L), one parental daphnid was considered pale on day 12. At 457 µg DMT(EHMA)/L, a maximum of 3 parental daphnids were pale on days 5 through 8, but were in good condition throughout the remainder of the test. At 2316 and 4879 µg DMT(EHMA)/L, respecitively, 10 and 9 parental daphnids were trapped at the surface and 9 and 10 parents were immobile on day 1. The one surviving parental daphnid at 2316 µg DMT(EHMA)/L was trapped at the surface on days 2,6 and 7, and considered small and pale on days 5-7. However, test solutions were not noted to contain a floating layer. The parental condition was considered treatment-related. It should be noted that no undissolved test substance or floating layer was observed in the test media.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The reproduction and body length data were not statistically analyzed, since reproduction and body lengths were higher in the treatment groups than in the control group.

Validity criteria fulfilled:

yes

Conclusions:

RESULTS CONTROL: 3 out of 20 parental daphnids died (15% mortality). Mortality occurred on days 19-21 of the study. On day 8, the first brood appeared. One daphnid aborted unhatched eggs.
Observed 21-day EC100 for parental survival: 2316 micrograms DMT(EHMA)/L
- 21-day LC50 for parental survival: 1029 micrograms DMT(EHMA)/L, no clear dose-response relationship was observed.
- Observed 21-day EC0 for parental survival: 457 micrograms DMT(EHMA)/L
- 21-day EC50 for reproduction: Could not be determined since reproduction among surviving daphnids was not affected
- 21-day LOEC for reproduction: 2316 micrograms DMT(EHMA)/L
- 21-day NOEC for reproduction: 457 micrograms DMT(EHMA)/L
- 21-day LOEC for body length: Could not be determined since body length among surviving daphnids was not affected
- 21-day NOEC for body length: 457 micrograms DMT(EHMA)/L
- Overall LOEC: 2316 micrograms DMT(EHMA)/L
- Overall NOEC: 457 micrograms DMT(EHMA)/L

Executive summary:

A Daphnia magna, reproduction test with dimethyltin (2 -ethylhexylthioglycolate). Fresh test solutions were prepared three times a week, staring with a stock at a loading rate of 100 mg/L. This stock solution was magentically stirred for 24 hours, after which it was left to stabilize for another 24 hours. The water fraction was then separated from the undissolved fraction of test substance using a pipette force.

In a semi-static design, ten daphnids per concentration were individually exposed to dilutions containing 1, 5, 10, 50, or 100% of the WAF prepared at 100 mg/L and twenty daphnids were indivdually expoased to untreated test medium (blank control).

Analytcal results were consistent within the groups and remained stable during the periods between media changes. Avergae exposure concnetrations were 49, 231, 457, 2316 and 4879 ug/L, corresponding to 1, 5, 10, 50 and 100% as WAF, respectively.

Treatment related deaths occurred only at the high concentrations of 2316 and 4879 ug/L. The six deaths amonth the other groups showed no dose response either by concentration or duration of exposure and could not be definitively ascribed to treatment. Three out of 20 (15%) of parental daphnids died in the control group and the deaths were clustered in the final three days of the days of the study. No cause could be determined for this and there was no dose-response, so the control deaths were attributed to coincidence and the validity of the conclusions for lethality and other endpoints measured in study was not compromised.

The average cumulative number of young per female in the control after 21 days was 100.8 +/-20.3. Reproduction at the three lowest treatment levels did not differ from the control. At the two highest test concentrations reproduction was completely inhibited due to the fact that all parents died before onset of reproduction.

Mean parental body length was not reduced up to 457 ug/L. At the two highest test concentrations, body length could not be determined since all had died.

Aborted eggs were observed in one control vessel on day 19, and in one vessel at 5% of the WAF on day 20. No further unhatched egggs or immobile young were observed at any concentrations tested. In the control and at the lowest treatment level, one parental daphnid was considered pale on day 12. At 457 ug/L, a maximum of 3 parental daphnids were pale on days 5 through 8, but were in good condition throughout the remainder of the test. At 2316 and 4879 ug/L, respectively, 10 and 9 parental daphnids were trapped at the surface and 9 and 10 parents were immobile on day 1. The one surviving parental daphnid at 2316 ug/L was trapped at the surface on days 2, 6 and 7, and considered small and pale on days 5 -7. However, test solutions were not noted to contain a floating layer. Hence, parental condition was considered treatment-related. It should be noted that no undissolved test substance or floating layer was observed in the test media.

Exposure to 2316 and 4879 ug/L induced significant inhibition of the reproductive capacity of the parental daphnids, due to the fact that no parents survived.

Observed 21-day EC100 for parental survival: 2316 micrograms DMT(EHMA)/L

- 21-day LC50 for parental survival: 1029 micrograms DMT(EHMA)/L, no clear dose-response relationship was observed.

- Observed 21-day EC0 for parental survival: 457 micrograms DMT(EHMA)/L

- 21-day EC50 for reproduction: Could not be determined since reproduction among surviving daphnids was not affected

- 21-day LOEC for reproduction: 2316 micrograms DMT(EHMA)/L

- 21-day NOEC for reproduction: 457 micrograms DMT(EHMA)/L

- 21-day LOEC for body length: Could not be determined since body length among surviving daphnids was not affected

- 21-day NOEC for body length: 457 micrograms DMT(EHMA)/L

- Overall LOEC: 2316 micrograms DMT(EHMA)/L

- Overall NOEC: 457 micrograms DMT(EHMA)/L

Description of key information

21-day NOEC 457 ug/L in Daphnia magna (OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.457 mg/L

Additional information

The long term toxicity to aquatic invertebrates was determined in a reproduction study in Daphnia magna following OECD test guideline 211. Exposure concentrations were as % water accommodated fractions of 1, 5, 10, 50 and 100% prepared at a loading rate of 100 mg/L (49, 231, 457, 2316 and 4879 ug/L as the test substance). Exposure to the two highest doses induced significant inhibition of the reproductive capacity of the parental daphnids, due to the fact that no parents survived.