Chlorotrioctylstannane

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

Study period:

09 July 2004 to 30 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Appearance: Slightly yellow liquid
- Density: 1.04 g/cm³
- Storage conditions of test material: In the freezer in the dark
- Stable under storage conditions: Yes
- Expiry date: 31 December 2004

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the study duplicate samples were taken from all test concentrations and the blank-control (0, 1, 5, 10, 50 and 100 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L).
- Sampling method: All test concentrations were sampled in duplicate at the beginning and the end of one interval of 72 hours (nominal days 0 and 3), and of three intervals of 48 hours (nominal days 5 and 7, 10 and 12, 17 and 19). The undiluted WAF was sampled additionally at the start of the second, fourth, sixth, seventh and ninth intervals (nominal days 3, 7, 12, 14 and 19). The sampling volume was 100 mL. In addition, singular reserve samples were taken from all test concentrations at these sampling times.
- Sample storage conditions before analysis: Stored at room temperature until transportation to site of analysis; once a week, all samples taken to site of analysis in 100 mL infusion bottles at room temperature. Reserve samples were stored at room temperature for possible analysis for a maximum of 3 months.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was considered insoluble (<0.1 mg/L at 20 °C) in test medium. 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 stabilise for another 24 hours. The water fraction was then separated from the undissolved fraction of test material using a pipette force. Finally, 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.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Age of parental stock (mean and range, SD):
- Feeding during test: Yes
- Food type: A suspension of fresh water algae (Chlorella pyrenoidosa). Algae suspensions were made by inoculating growth medium with Chlorella pyrenoidosa from a pure culture. The suspensions were continuously aerated and exposed to light (6000 to 10 000 lux; 60-120 µE/m²/s) in a climate room at a temperature of 23 ± 2 °C for 2 to 3 weeks.
- Amount: A ratio of ca. 0.2 mg C/daphnid/day
- Frequency: Daily

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 L of medium
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week
- Feeding: Daily, a suspension of Chlorella pyrenoidosa
- Validity of batch: Frequent inspection with respect to number of young, and appearance of young and parental daphnids
- Medium: M7

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

268 to 357 mg/L CaCO3

Test temperature:

19.3 to 20.3 °C

pH:

7.5 to 8.7

Dissolved oxygen:

6.0 to 10. 5 mg/L

Nominal and measured concentrations:

- Nominal test concentrations: A WAF prepared at a loading rate of 100 mg/L and dilutions containing 1, 5, 10 and 50 % of the WAF were prepared.
- Measured: the average concentrations were 0.057, 0.11, 0.073, 0.14, 0.48 and 1.4 µg/L for the blank, 1, 5, 10, 50 and 100 % solutions, respectively. The lower concentrations (in WAFs up to 5 %) were considered to approximate the detection limit.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass. Test vessels were pre-incubated with the respective test solution for 30 minutes.
- Renewal rate of test solution (frequency/flow rate): Three times a week
- Experimental design: From the start of the experiment (nominal day 0) 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 mL test medium. The control group consisted of 20 daphnids.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium (ISO-medium 6341); the following chemicals are dissolved in freshly prepared ultra-pure water (tap water) purified by reverse osmosis (milli-RO): 293.8 mg/L CaCl2.2H2O, 123.3 mg/L MgSO4.7H2O, 64.8 mg/L NaHCO3 and 5.8 mg/L KCl.
Trace elements, macro nutrients and vitamins are added to freshly prepared ISO-medium.
Trace elements: 0.125 mg/L B, 0.05 mg/L Fe, 0.025 mg/L Mn, 0.0125 mg/L Li, Rb and Sr, 0.0063 mg/L Mo, 0.0025 mg/L Br, 0.0016 mg/L Cu, 0.0063 mg/L Zn, 0.0025 mg/L Co and I, 0.0010 mg/L Se, 0.0003 mg/L V and 2.5 mg/L Na2EDTA.2H2O.
Macro nutrients: 10.0 mg/L Na2SiO3.9H2O, 0.27 mg/L NaNO3, 0.14 mg/L KH2PO4 and 0.18 mg/L K2HPO4.
Vitamins: 75 µg/L Thiamine, 1.0 µg/L B12 and 0.75 µg/L Biotin.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen were measured at the start of the test and just before and after each renewal in all test solutions. Additionally, temperature was monitored continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h photoperiod daily
- Light intensity: between 610 and 793 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
Parental Daphnia
- Immobility and mortality: Every workday, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
- Presence of eggs in the brood pouch: Every workday
- Body length: At the end of the test
F1 Generation
- Appearance first brood: When observed
- Newborn daphnids: Every workday the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
- Presence of unhatched eggs: When observed
- Incidence of immobility: When observed

RANGE-FINDING STUDY: Yes
The test material was expected to be slightly toxic to Daphnia magna. According to a pre-test, the acute EC50 approximated the concentration obtained in a WAF prepared at a loading rate of 100 mg/L. It should be noted that in this pre-test, the WAF was not filtered, and relatively high numbers of floating daphnids (up to 100 %) were observed.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: parental survival

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: parental survival, reproduction and body length

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: parental survival, reproduction and body length

Details on results:

MEASURED CONCENTRATIONS
The analytical data do not confirm the repeatability of the preparation of the test concentrations and the stability of the concentrations during the periods between renewals. The variation in initial concentrations measured in the stock solution is attributable to the character of the test material (including low water solubility). Tin was not detected in control samples, except in old solutions on day 7, and in one sample from the old solution on day 12. The measured concentrations in old solutions on day 7 were not in line with what was expected based on the other intervals. The concentrations measured in old medium decreased with increasing percentage of the WAF, which was not seen in other intervals. Moreover, initial concentrations in this interval were in general higher than in the other intervals. There is no explanation for this, other than the extremely low water solubility of the test substance and the difficulties encountered during separation of the water soluble fraction. Since measured concentrations in this interval clearly deviated from the general trend in the entire test period, this interval was excluded from calculations.
Note that the average exposure concentrations were calculated from the data of intervals where both the initial and the final concentration were measured, i.e. the 1st, 5th and 8th interval. The measured concentrations of the fresh solutions of the 2nd, 4th, 6th, 7th and 9th interval were only used to verify the repeatability of the preparation of test solutions.

PARENTAL MORTALITY
Cumulative mortality is shown in Table 2. There was no clear dose response in mortality, except for the highest concentration of 1.4 µg test material/L. Based on these results, the 21-day LC50 was 1.0 µg test material/L.
One out of the twenty (5 %) parental daphnids died during the test period in the control.

REPRODUCTION
The first young in the control were observed on day 10, but this may have been an artefact of the observation, since offspring were not recorded on days 8 and 9. The average cumulative number of young per reproducing female in the control after 21 days was 126.1 ± 15.0. The reproduction curves recorded up to 0.48 µg test material/L (50 % WAF) closely followed or slightly exceeded the curve of the control. At the highest treatment, 1.4 µg test material/L, reproduction was significantly reduced.

BODY LENGTH
Mean body length was not significantly reduced up to 10 % WAF (0.14 µg test material/L), but was significantly reduced at the two highest tested concentrations of 0.48 and 1.4 µg test material/L (Bonferroni t-test and Tukey test, α=0.05).

OTHER PARAMETERS
No unhatched eggs or immobile offspring were observed in any of the concentrations tested.
One immobile parental daphnid was observed in the control on day 20. This parent had died on day 21. At 1 % WAF, 3 parental daphnids were smaller and did not have a brood pouch during the study period. These daphnids were considered to be males and were not included in calculations of reproduction. At all higher treatment levels, one daphnid was small and did not develop a brood pouch. These were treated similarly as those in the lowest test concentration. At the highest concentration, 100 % WAF (1.4 µg test material/L), one parental daphnid was immobile on day 18; this daphnid was found dead on day 19. Another daphnid was immobile on day 21 (the other deaths occurred without preceding bad condition of parents). Hence, poor parental condition (concerning sub-lethal effects) was not considered to be treatment-related.

LOEC, NOEC, EC and LC VALUES
The LC50 for survival in parental daphnids was estimated to be 81 %. The observed 21-day LOEC for parental survival was 100 % of a WAF prepared at a loading rate of 100 mg/L and the EC0 for parental survival was estimated at 50 % of the WAF.
The 21-day LOEC for reproduction was 100 % of a WAF prepared at a loading rate of 100 mg/L, while the 21-day NOEC for reproduction was 50 %. The 21-day EC50 for reproduction was >100 %.
The 21-day LOEC for body length was 50 % of a WAF prepared at 100 mg/L, while the NOEC was 10 % of the same WAF.
Hence, the overall LOEC and NOEC were 50 and 10 % of a WAF prepared at 100 mg/L, respectively.

Reported statistics and error estimates:

The values for reproduction observed at various concentrations of the test substance in water were expressed as mean number of living young per parent.

For each concentration, the results of reproduction were tested for normality and for homogeneity of variance. Furthermore, these data were statistically tested using an ANOVA test followed by a mean comparison test (Bonferroni t-test, Tukey test and Dunnett's test; SAS program release 6.12). The Debtox model was used to calculate the EC50 for reproduction.
The LC50 was determined using the maximum likelihood estimation method with the probits of the percentages of dead daphnia as function of the logarithms of the corresponding concentrations (Finney 1971).
Body length was statistically tested using an ANOVA test followed by a Bonferroni t-test and a Tukey test (TOXSTAT Release 3.5).
The overall threshold level of effect and the overall NOEC were determined on basis of these statistics. The LC50 (parental survival) and the EC50 (reproduction) were calculated.

Table 1: Analytical results (concentrations in µ/L)

Interval	Nominal day	Date	Test Solution	% of a WAF at 100 mg/L
				1	5	10	50	100
1	Day 0	09/072004	Fresh	0.05*	0.05*	0.14	0.49	1.0
	Day 3	12/07/2004	Old	0.085	0.080	0.19	0.21	0.37
	0-3		Mean	0.065	0.063	0.16	0.32	0.61
2	Day 3	12/07/2004	Fresh					3.3
3	Day 5	14/07/2004	Fresh	0.27	0.56	1.2	5.5	14
	Day 7	16/07/2004	Old	1.2	0.29	0.12	0.11	0.05*
	5-7		Mean	0.56**	0.40**	0.37**	0.79**	0.84**
4	Day 7	16/07/2004	Fresh					0.90
5	Day 10	19/07/2004	Fresh	0.05*	0.15	0.29	1.1	7.3
	Day 12	21/07/2004	Old	0.05*	0.05*	0.08	0.47	1.3
	10-12		Mean	0.05*	0.087	0.15	0.72	3.0
6	Day 12	21/07/2004	Fresh					1.3
7	Day 14	23/07/2004	Fresh					1.4
8	Day 17	26/07/2004	Fresh	0.25	0.095	0.23	0.46	0.76
	Day 19	28/07/2004	Old	0.19	0.05*	0.05*	0.38	0.44
	17-19		Mean	0.22	0.069	0.11	0.42	0.57
9	Day 19	28/072004	Fresh					1.0
Average exposure	0-21		Mean	0.11	0.073	0.14	0.48	1.4

*Concentration below LOD, being 0.10 µg/L; tabled value is half the LOD

**Not included in calculation of average exposure concentrations

Table 2: Cumulative mortality of parental daphnids during the 21-day exposure period

Test Material Conc. (% of 100 mg/L WAF)	Exposure (µg/L)	Number Exposed	Cumulative number of dead parental daphnids on day:															Mortality %
			3	4	5	6	7	10	11	12	13	14	17	18	19	20	21
Blank	0.057	20	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	5
1	0.11	10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5	0.073	10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
10	0.14	10	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	10
50	0.48	10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
100	1.4	10	0	0	0	0	0	0	0	0	0	0	1	2	4	6	8	80

Table 3: LOEC, NOEC, EC and LC Values

Parameter	Percentage of 100 mg/L WAF	Exposure concentration (µg/L)
		Test Material
Observed 21-day EC100 for parental survival	>100	>1.4
21-day LC50 for parental survival¹	81	1.0
Observed 21-day EC0 for parental survival	50	0.48
21-day EC50 for reproduction²	>100	>1.4
21-day LOEC for reproduction³	100	1.4
21-day NOEC for reproduction³	50	0.48
21-day LOEC for body length⁴	50	0.48
21-day NOEC for body length⁴	10	0.14
Overall LOEC	50	0.48
Overall NOEC	10	0.14

¹Finney

²Estimated by Debtox

³ANOVA - Dunnet T-test, Bonferroni T-test and Tukey’s test; α = 0.05

⁴ANOVA - Bonferroni T-test and Tukey’s test, α = 0.05

Validity criteria fulfilled:

yes

Conclusions:

The 21-day LC50 for survival in parental daphnids was 1.0 µg test material/L; the overall LOEC and NOEC were 0.48 and 0.14 µg test material/L, respectively.

Executive summary:

A study was conducted to investigate the effect of the test material on the mortality and reproduction of Daphnia magna over a 21-day period in accordance with the standardised guideline OECD 211 under GLP conditions.

In a semi-static design, ten daphnids per concentration were individually exposed to dilutions containing 1, 5, 10, 50 or 100 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L, and twenty daphnids were individually exposed to untreated test medium. The test material was considered insoluble (<0.1 mg/L at 20 °C) in test medium at the concentrations tested. Test solutions were renewed three times a week and fresh test solutions were prepared three times a week, starting with a stock at a loading rate of 100 mg/L.

Samples taken from the vessels were analysed for tin concentration. Thus, the concentration can be reported as % WAF or as µg/L test material. The latter is based on the assumption that all measured tin is the test material. Analytical results were consistent within the groups and remained stable during the periods between media changes. Reported exposure concentrations for each group were calculated by taking the mean of analytical results of whole intervals for the group. Thus, average exposure concentrations were 0.11, 0.073, 0.14, 0.48, and 1.4 µg/L at 5, 10, 50, and 100 % WAF, respectively. The 1, 5 and 10 % WAF samples contained concentrations below the limit of detection (LOD) and are reported as one half the LOD or 0.05 µg test material/L.

Treatment-related deaths were observed at the highest test concentration. One parental daphnid died in the control group. At 10 and 100 % of the WAF (0.14 and 1.4 µg test material/L), 1 and 8 daphnids died during the test.

The average cumulative number of young per reproducing female in the control after 21 days was 126.1 ± 15.0. The reproduction curves recorded up to 0.48 µg test material/L (50 % WAF) closely followed or slightly exceeded the curve of the control. At the highest treatment (1.4 µg/L), reproduction was significantly reduced.

Mean body length was not significantly reduced up to 10 % WAF (0.14 µg/L), but was significantly reduced at the two highest tested concentrations of 0.48 and 1.4 µg/L.

No unhatched eggs or immobile offspring were observed in any of the concentrations tested.

The 21-day LC50 for survival in parental daphnids was 1.0 µg test material/L; the overall LOEC and NOEC were 0.48 and 0.14 µg test material/L, respectively.

Description of key information

21 d LC50 (survival in parental daphnids) = 1.0 µg/L. The overall LOEC and NOEC were 0.48 and 0.14 µg/L, respectively, OECD 211, de Roode & de Haan (2004)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.14 µg/L

Additional information

A study was conducted to investigate the effect of the test material on the mortality and reproduction of Daphnia magna over a 21-day period in accordance with the standardised guideline OECD 211 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

In a semi-static design, ten daphnids per concentration were individually exposed to dilutions containing 1, 5, 10, 50 or 100 % of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L, and twenty daphnids were individually exposed to untreated test medium. The test material was considered insoluble (<0.1 mg/L at 20 °C) in test medium at the concentrations tested. Test solutions were renewed three times a week and fresh test solutions were prepared three times a week, starting with a stock at a loading rate of 100 mg/L.

Samples taken from the vessels were analysed for tin concentration. Thus, the concentration can be reported as % WAF or as µg/L test material. The latter is based on the assumption that all measured tin is the test material. Analytical results were consistent within the groups and remained stable during the periods between media changes. Reported exposure concentrations for each group were calculated by taking the mean of analytical results of whole intervals for the group. Thus, average exposure concentrations were 0.11, 0.073, 0.14, 0.48, and 1.4 µg/L at 5, 10, 50, and 100 % WAF, respectively. The 1, 5 and 10 % WAF samples contained concentrations below the limit of detection (LOD) and are reported as one half the LOD or 0.05 µg test material/L.

Treatment-related deaths were observed at the highest test concentration. One parental daphnid died in the control group. At 10 and 100 % of the WAF (0.14 and 1.4 µg test material/L), 1 and 8 daphnids died during the test.

The average cumulative number of young per reproducing female in the control after 21 days was 126.1 ± 15.0. The reproduction curves recorded up to 0.48 µg test material/L (50 % WAF) closely followed or slightly exceeded the curve of the control. At the highest treatment (1.4 µg/L), reproduction was significantly reduced.

Mean body length was not significantly reduced up to 10 % WAF (0.14 µg/L), but was significantly reduced at the two highest tested concentrations of 0.48 and 1.4 µg/L.

No unhatched eggs or immobile offspring were observed in any of the concentrations tested.

The 21-day LC50 for survival in parental daphnids was 1.0 µg test material/L; the overall LOEC and NOEC were 0.48 and 0.14 µg test material/L, respectively.