2-ethylhexyl 12-ethyl-5,5-dioctyl-9-oxo-10-oxa-4,6-dithia-5-stannahexadecanoate

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

December 4th 2003 to September 6th 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Reliability of original study is 1

Justification for type of information:

Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

other: report amendment

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 3

Reference Type:

other: report amendment

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

239-622-4

EC Name:

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

Cas Number:

15571-58-1

Molecular formula:

C36H72O4S2Sn

IUPAC Name:

2-ethylhexyl 10-ethyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- During the study duplicate samples were taken from all test concentrations and the blank-control.
- Sampling frequency: all test concentrations: in duplicate at the beginning and the end of three intervals of 48 hours (nominal days 0 and 2, 5 and 7, and 12 and 14) and of an interval of 72 hours (nominal days 16 and 19). Undiluted WAF: additionally at the start of the 5th, 7th and 9th interval (nominal days 9, 14 and 19).
- Sample volume: 25 ml.
- Treatment: 50 ml of acetic acid (100 %, Merck) was added to each sample directly after sampling.
- Storage: samples were stored at room temperature until transportation to the laboratory.
- Transport: once every week, all samples taken were sent to the laboratory, in 100 ml polyethylene bottles at room temperature.
- In addition, singular reserve samples were taken from all test concentrations at the days mentioned above. These samples were not treated with acetic acid or sent to the laboratory. If not already uses, these samples were stored at room temperature for possible analysis for a maximum of 3 months after delivery of the draft report pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS: the standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. Concentrations that disturb the test system were excluded from testing (e.g. film of the test substance on the water surface or deposits of test substance on the bottom of the vessels). 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 substance 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 concentrations annotated as 1 %, 5 %, 10 %, 50 % and 100 % contained 1, 5, 10, 50 and 100 % of the prepared WAF, respectively. Test vessels were preincubated with the respective test solutions for 1/2 hour.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820).
- Reason for selection: this system has been selected as an internationally accepted species.
- Age: young daphnids with an age of < 24 hours.
- Feeding during test: daily, a quantity of Chlorella pyrenoidosa suspension was added as feed for the daphnids providing a ration of ca. 0.2 mg C/daphnid/day.

BREEDING
- Start of each batch: with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres 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).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

268-304 mg CaCO3/l

Test temperature:

19.4-20.8 °C (test media) and 18.7-20.8 °C (temperature of room). Temperature was maintained within the limits prescribed by the protocol, except for four short periods when the room temperature, measured in the temperature control vessel, was out of range.

pH:

7.6-8.6

Dissolved oxygen:

> 6.0 mg/l

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal: 1, 5, 10, 50 and 100 % of a WAF prepared at a loading rate of 100 mg/l.
Average measured: 33, 134, 286, 1448, and 3213 μg/l (associated with the 0 mg/l blank and the 1, 5, 10, 50, and 100 % of the WAF prepared at 100 mg/l, respectively).

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels.
- Fill volume: 50 ml.
- No. of organisms in test solutions: 10 daphnids per group were divided over ten vessels.
- No. of organisms in control: 20.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 as prescribed by Dr. Elendt-Schneider (1990).
- Water parameters measurement: temperature and pH was measured at the start of the test and just before and after each renewal in all test solutions. The temperature was additionally measured continuously in a temperature control vessel. For T(D)OC measurements: samples (duplicate, 10 ml) for measurement of total (dissolved) organic carbon were taken on day 0. T(D)OC analyses, apparatus: analyses were performed using a DC-190 High Temperature Total organic Carbon Analyzer.

OTHER TEST CONDITIONS
- Photoperiod: 16 h daily.
- Light intensity: 620 and 798 lux.

CONTROL: test medium without test substance or other additives (0 mg/l).

EFFECT PARAMETERS MEASURED
- Parental Daphnia
- Immobility and mortality: every workday, the number of living, immobile and dead parental daphnids were 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.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: the test material was not expected to be very toxic to Daphnia magna. According to a pretest, the acute EC50 was expected above the concentration obtained in a WAF prepared at a loading rate of 100 mg/l.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 3 213 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: parental survival

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 3 213 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3 213 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 448 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

617 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction: mobile offspring

Effect concentrations 6

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 448 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction: mobile offspring

Effect concentrations 7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

286 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction: mobile offspring

Effect concentrations 8

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 448 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: body length

Effect concentrations 9

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

286 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: body length

Effect concentrations 10

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 448 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: overall LOEC: parental survival, reproduction, mobility of offspring, and body length

Effect concentrations 11

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

286 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: overall NOEC: parental survival, reproduction, mobility of offspring and body length

Details on results:

- Mortality of parent animals: none of the twenty parental daphnids died during the test period in the control. Incidental mortalities occurred at all but the lowest treatment level, with 1, 1, 2 and 2 dead parents at 134, 286, 1448 and 3213 µg/l, respectively. Hence, parental mortality was not related to concentration. Based on these results, the LC50 was above the maximum concentration tested. EC0 = 286.0 μg/l (measured-arithm. mean) parental survival; EC100 > 3213.0 μg/l (measured-arithm. mean) parental survival).

- No. of offspring produced per day per female: the reproduction curves recorded at the all treatment levels closely followed the curve of control. However, at the two highest test concentrations, offspring included considerable numbers of immobile young. After exclusion of these immobile daphnids, the reproduction curves recorded at the two highest treatment levels were much lower than the control.
Statistical analysis on the time curves for cumulative reproduction of young (total production) showed that the reproduction of the daphnids was significantly reduced at the highest test concentration (Bonferroni test, Tukey test and Dunnett test, ct=0.05).
Statistical analysis on the time curves for cumulative reproduction of mobile young showed that the reproduction of the daphnids was significantly reduced at the two highest test concentrations (Bonferroni test, Tukey test and Dunnett test, a=0.05).

- Body length and weight of parent animals: mean body length was statistically reduced at the highest two concentrations (Bonferroni t-test and Tukey test, a=0.05).

- Other biological observations: no unhatched eggs or dead offspring were observed in any of the concentrations tested. No small or pale parental daphnids were observed in the control or the test concentrations throughout the test period.

Reported statistics and error estimates:

STATISTICAL ANALYSIS: 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 for Windows NT). The Debtox model (M. Luger, J. Bedeaux and B. Kooijman, 1998, the Netherlands) 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 daphnids as function of the logarithms of the corresponding concentrations (Finney, DJ., 1971: Probit analysis, Cambridge University Press, Cambridge, u.K., 3rd edition).

Body length was statistically tested using an ANOVA test followed by a Bonferroni t-test and a Tukey test (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 basis of these statistics. The LC50 (parental survival) and the EC50 (reproduction) were calculated.

Any other information on results incl. tables

Table: cumulative mortality of parental Daphnids during the 21-day exposure period

Concentration^1of the substance	Exposure (µg/l)	Number exposed	Cumulative number of dead parental daphnids on day:															Mortality %
			1	2	5	6	7	8	9	12	13	14	15	16	19	20	21
0 mg/l (Blank)	0	20	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1 %	33	10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5 %	134	10	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	10
10 %	286	10	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	10
50 %	1448	10	0	0	0	0	0	0	0	1	1	2	2	2	2	2	2	20
100 %	3213	10	0	0	0	0	0	0	0	0	0	0	0	0	1	2	2	20

Table: cumulative mean number of mobile young per parent as percentage of the mean number of total young per parent at the various treatments (based on loading rates of the test substance in mg/l) during the 21 day test period.

Concentration^1 of the substance	Cumulative number of mobile young as a percentage of total young per parent
	8	9	12	13	14	15	16	19	20	21
0 mg/l (Blank)	100	100	100	100	100	100	100	100	100	100
1 %	-	100	100	100	100	100	100	100	100	100
5 %	-	100	100	100	100	100	100	100	100	100
10 %	-	100	93	94	95	96	97	98	95	95
50 %	-	56	25	23	15	16	20	30	30	35
100 %	-	14	4.2	3.8	2.7	2.1	4	9.8	7.1	14

Table: average bodylengths (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 control.

Concentration^1 of the substance	Exposure in µg/l	Average length (mm)	SD	% reduction
0 mg/l (Blank)	0	4.8	0.08
1 %	33	4.7	0.07	2.1
5 %	134	4.7	0.06	2.1
10 %	286	4.7	0.1	2.1
50 %	1448	4.6*	0.08	4.2
100 %	3213	4.4*	0.09	8.3

¹% of a WAF at 100 mg/l.

*Significantly different from control at the 5 % level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure to 3213 µg/l induced significant inhibition of the reproductive capacity of the parental daphnids.
Based on mobile offspring, the highest two treatment concentrations, 1448 and 3213 µg/l significantly reduced the reproductive capacity.

Executive summary:

The long-term toxicity potential of the test material was evaluated in a semi-static test according to the guideline OECD211. Ten daphnids per concentration were individually exposed to dilutions containing 1, 5, 10, 50 and 100 % of the WAF prepared at 100 mg/l, and twenty daphnids were individually exposed to untreated test medium (blank control). Test solutions were renewed three times a week. The total test period was 21 days. Samples for analysis were taken from all test solutions at the start and the end of the first, third, sixth and eighth renewal interval. Additionally, samples were taken from the freshly prepared highest test solution at the start of the fifth, seventh and ninth interval. 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. The exposure test material concentrations were: 33, 134, 286, 1448 and 3213 µg/l. Considering the extremely low solubility of the test substance super-saturated solutions were prepared. In this context tested concentrations expressed as WAF are far above the water solubility of the test substance. Therefore the effects noticed cannot necessarily be attributed to parent compound.

None of the twenty parental daphnids died during the test period in the control. Incidental mortalities occurred at all but the lowest treatment level, with 1, 1, 2 and 2 dead parents at 134, 286, 1448 and 3213 µg/l, respectively. Hence, parental mortality was not related to concentration. EC50 (21 d) > 3213.0 μg/l (measured-arithm. mean).

The average cumulative number of young per reproducing female in the control after 21 days was 134.6 ± 14.2. The reproduction curves recorded at all the treatment levels closely followed the curve of control. However, at the two highest test concentrations, 1448 and 3213 µg/l, offspring included considerable numbers of immobile young. The number of mobile offspring at the two highest treatment levels was statistically significantly reduced. Based on total offspring produced, only the highest treatment level showed a statistically significant reduction.

Mean body length was significantly reduced at 1448 and 3213 µg/l.

No unhatched eggs were observed in any of the concentrations tested. No small or pale parental daphnids were observed in the control or the test concentrations throughout the test period.