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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 December 2003 to 03 December 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted under GLP. Test solutions were analyzed on the basis of total tin and were converted to and reported as the test substance, MOT(2-EHMA). The preparation of the Water Accomodated Fraction (WAF) allowed both the soluble portion of the test substance and the hydrolysis products to be present in the medium for the chronic study. For the 10% WAF half of the samples were below the LOD (4.5 µg/L). At 1% and 5%, all samples were below the LOD. For the concentrations below the LOD, half the LOD (2.3 µg/L) was used to calculate the Sn concentration. Additionally, purity of test substance was only 54.0%. Concerns regarding the substances composition in particular the presence of the impurity EHTG mean that this study was used as a worst case scenario with regard to risk assessment but is not used for classification and labelling which is based on the newer and more reliable acute toxicity studies performed on a purer version of the substance. Further investigation of this end point is due to be performed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
Minor deviations from the OECD 211 protocol in test solution preparation and statistical analysis, these deivations were considered not to have affected the reliability of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS
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 2nd, 4th, 5th, 7th and 9th interval (nominal days 2, 7, 9, 14 and 19).
Sample volume: 25 ml.
Treatment: 50 ml of acetic acid (Merck, 100%) was added to each sample directly after sampling.
Storage: Samples were stored at room temperature until transportation to TNO Nutrition and Food Research.
Transport: Once-twice every week, all samples taken were sent to TNO Nutrition and Food Research, 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 TNO. If not already useD, 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.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
Characteristics: For the test selection of young daphnids with an age of < 24 hours.

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)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
250-304 mg CaCO3/L

Test temperature:
19.0-20.8°C (test media) and 18.2-20.1°C (temperature of room). Temperature was maintained within the limits prescribed by the protocol. Temperature was measured at the start of the test and just before and after each renewal in all test solutions. Additionally, continuously in a temperature control vessel.
pH:
6.7-8.3 (maintained within the limits prescribed by the protocol); measured at the start of the test and just before and after each renewal in all test solutions.
Dissolved oxygen:
>6.0 mg/L, in compliance with protocol; measured at the start of the test and just before and after each renewal in all test solutions.
Salinity:
NA
Nominal and measured concentrations:
% of a WAF at 100 mg/l: 1, 5, 10, 50 and 100 [nominal]

Average exposure concentration in µg Sn/l: 2.3*, 2.7, 5.0, 22 and 49. * Concentration below LOD being 4.5 µg/l - value is half the LOD
Average exposure concentration in µg MOT(EHMA)/l: 16, 19, 36, 157 and 344



Controls: Test medium without test substance or other additives (0 mg/L).

The average measured concentrations of the test substance, used as the average exposure concentrations for each test, were 0 mg/L in the blank and 16, 19, 36, 157, and 344 ug/L MOT(EHMA) (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:
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).

The batch of MOT(EHMA) tested was a colourless liquid with a purity of 54.0% and not completely soluble in test medium. The method of preparation was based on NOTOX Project 374941. 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 at least 30 minutes.

TEST PROCEDURE AND CONDITIONS
Frequency of renewal: Three times a week
Test duration: 21 days
Test vessels: All-glass
Medium: M7 as prescribed by Dr. Elendt-Schneider.
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.
Light: 16 h photoperiod daily, intensity between 535 and 730 lux.
Feeding: 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.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
ca. 228 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
other: parental survival
Duration:
21 d
Dose descriptor:
other: EC0
Effect conc.:
ca. 36 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
other: parental survival
Duration:
21 d
Dose descriptor:
other: EC100
Effect conc.:
> 344 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
other: parental survival
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 36 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on total offspring (mobile or not)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
157 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on total offspring (mobile or not)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 344 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on total offspring (mobile or not)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
36 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on mobile offspring
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
157 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on mobile offspring
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
225 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
reproduction
Remarks:
based on mobile offspring
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
157 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
morphology
Remarks:
body length
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
344 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
morphology
Remarks:
body length
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
36 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
other: overall
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
157 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
- measured tin and converted to MOT(EHMA)
Basis for effect:
other: overall
Reported statistics and error estimates:
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."

PARENTAL MORTALITY

No parental daphnids died during the test period in the control. One incidental mortality (one out often

exposed daphnids) occurred at 1% of the WAF. At 50% and 100% of the WAF prepared at a loading rate of 100 mg/L, 3 and 7 daphnids died during the test,

respectively. Hence, parental mortality was related to concentration. The LC50 was

calculated using Finney.


REPRODUCTION

The reproduction curves recorded at all but the highest treatment level closely followed the curve of the control. However, at 50% of the WAF, 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.

The control showed a rather high variation in reproduction, with a standard deviation of 44% of the mean. This was mainly caused by the presence of three daphnids that were considered small and produced significantly less young than the rest throughout the test period. Exclusion of these parents reduced the coefficient of variation to 25%. This is still slightly higher than allowed by the guideline. However, since there was no indication that reproduction was negatively affected at the three lowest treatment levels, this deviation was accepted.

Statistical analysis on the total reproduction of young (total production) showed that the reproduction of the daphnids was significantly reduced at 50 and 100% of the WAF (157 and 344 µg MOT(EHMA)/L); Bonferroni test and Tukey test and

Dunnett test, α=0.05).

Statistical analysis on the reproduction of mobile young showed that the reproduction of the daphnids was significantly reduced at the two highest test concentrations of 50 and 100% of the WAF (157 and 344 µg MOT(EHMA)/L; Bonferroni test and Tukey test and Dunnett test, α=0.05).

BODY LENGTH

Mean body length was statistically reduced at the highest concentration of 100% of the WAF (344 µg MOT(EHMA)/L; Bonfenoni t-test and Tukey test, α=0.05).

Concentration MOT(EHMA) Exposure in µg/L Average length (MM) SD % reduction
0 0.0 4.3 0.320 -
1% 16.0 4.2 0.220 2.3
5% 19.0 4.3 0.240 0
10% 36.0 4.3 0.200 0
50% 157.0 4.3 0.280 0
100% 344.0 3.8 0.340 12

OTHER PARAMETERS

Aborted eggs were observed in the control and all concetrations tested except for the highest concentration. The incidence was not related to treatment. Immobile young were seen incidentally in the control and the lowest concentration.In both cases, only one parent produced 3 or 6 immobile young. These parents were observed to be in poor condition throughout the test and not included in calculations or statistical analysis. Fmther, significant numbers of immobile young were produced at 50% of the WAF (157 µg MOT(EHMA)/L).

Parental condition was not clearly related to treatment, with small and pale

daphnias in all concentrations and the control. However, there was a dose dependent increase in the number of daphnia trapped at the surface, even

though a test substance film or floating layer was not observed.

Validity criteria fulfilled:
yes
Conclusions:
This study investigated the effects of MOT(EHMA) on survival and reproductive performance in Daphnia magna during 21 days of exposure. Exposure to 157 and 344 µg MOT(EHMA)/L induced significant inhibition of the reproductive capacity of the parental daphnids. Overall LOEC = 157µg MOT(EHMA)/L. Overall NOEC = 36 µg/L.
Executive summary:

Daphnia magna, reproduction test with mono-octyltin tris(2-ethylhexylmercaptoacetate) (MOT(EHMA)). The study procedures described in this report were based on the OECD guideline No. 211: " Daphnia magna, Reproduction Test", Adopted: 21st September 1998.

The method of preparation was based on NOTOX Project 374941 (non-GLP study). 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.

In a semi-static design, ten daphnids per concentration were exposed to dilutions containing 1, 5, 10, 50 or 100% of the WAF prepared at 100 mg/L, and twenty daphnids were 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 second, fourth, fifth, seventh and ninth interval.

The samples taken from the vessels were stabilized and diluted with acetic acid and then analysed for Sn concentration. Thus, the concentration can be reported in three ways: as % WAF, as µg Sn/L, or as µg MOT(EHMA)/L. The latter is calculated based on the molecular weight ratio of MOT(EHMA) and Sn and assumes all measured Sn originated from the test substance.

Analytical results were consistent within the groups, but concentrations did not remain 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 as follows:

As WAF: 1.0, 5.0, 10, 50, and 100%

As Sn: 2.3, 2.7, 5.0, 22, and 49 µg/L

As MOT(EHMA): 16, 19, 36, 157, and 344 µg/L

The 1, 5 and 10% WAF samples contained concentrations below the limit of detection (LOD) and are reported as one half the LOD or 2.3 µg Sn/L and 16 µg MOT(EHMA)/L in the report.

Treatment-related deaths occurred at the concentrations of 157 and 344 µg MOT(EHMA)/L. The one death in the other groups (16 µg MOT(EHMA)/L) showed no dose-response either by concentration or duration of exposure and could not be definitively be ascribed to treatment. No deaths occurred in the control group.

The average cumulative number of young per reproducing female in the control after 21 days was 128.7 ± 32.2. Reproduction at 16, 157 and 344 µg MOT(EHMA)/L was significantly reduced. Since reproduction at 19 and 36 µg MOT(EHMA)/L was not reduced, the NOEC was considered to be 36 µg MOT(EHMA)/L. At the test concentrations of 157 and 344 µg MOT(EHMA)/L, production of mobile young was significantly reduced.

Mean parental body length was not significantly reduced at concentrations up and including to 157 µg MOT(EHMA)/L. At the highest test concentration (344 µg MOT(EHMA)/L), body length was reduced by 12% compared to the control. This was considered a treatment related outcome.

Aborted eggs were observed in the control and all but the highest concentration tested. The incidence was not related to treatment. Immobile young were seen incidentally in the control and the lowest concentration. In both cases, only one parent produced 3 or 6 immobile young. These parents were observed to be in poor condition throughout the test and not included in calculations or statistical analysis. Further, significant numbers of immobile young were produced at 50% of the WAF (157 µg MOT(EHMA)/L).

Parental condition was not clearly related to treatment, with small and pale daphnias in all concentrations and the control. However, there was a dose dependent increase in the number of daphnia trapped at the surface, even though a test substance film or floating layer was not observed.

Test conditions for pH, temperature, dissolved oxygen, hardness and TOC remained within the limits prescribed by the protocol and the test was considered valid.

For the lethality endpoint, LC50 was calculated to be 228 µg MOT(EHMA)/L; the overall 21-day LOEC was 157 µg MOT(EHMA)/L and the overall NOEC was 36 µg MOT(EHMA)/L.

Description of key information

The 21 -day NOEC was 36 µg MOT(EHMA)/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
36 µg/L

Additional information

Daphnia magna, reproduction test with mono-octyltin tris(2-ethylhexylmercaptoacetate) (MOT(EHMA)). The study procedures described in this report were based on the OECD guideline No. 211: " Daphnia magna, Reproduction Test", Adopted: 21st September 1998.

The method of preparation was based on NOTOX Project 374941 (non-GLP study). 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.

In a semi-static design, ten daphnids per concentration were exposed to dilutions containing 1, 5, 10, 50 or 100% of the WAF prepared at 100 mg/L, and twenty daphnids were 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 second, fourth, fifth, seventh and ninth interval.

The samples taken from the vessels were stabilized and diluted with acetic acid and then analysed for Sn concentration. Thus, the concentration can be reported in three ways: as % WAF, as µg Sn/L, or as µg MOT(EHMA)/L. The latter is calculated based on the molecular weight ratio of MOT(EHMA) and Sn and assumes all measured Sn originated from the test substance.

Analytical results were consistent within the groups, but concentrations did not remain 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 as follows:

As WAF: 1.0, 5.0, 10, 50, and 100%

As Sn: 2.3, 2.7, 5.0, 22, and 49 µg/L

As MOT(EHMA): 16, 19, 36, 157, and 344 µg/L

The 1, 5 and 10% WAF samples contained concentrations below the limit of detection (LOD) and are reported as one half the LOD or 2.3 µg Sn/L and 16 µg MOT(EHMA)/L in the report.

Treatment-related deaths occurred at the concentrations of 157 and 344 µg MOT(EHMA)/L. The one death in the other groups (16 µg MOT(EHMA)/L) showed no dose-response either by concentration or duration of exposure and could not be definitively be ascribed to treatment. No deaths occurred in the control group.

The average cumulative number of young per reproducing female in the control after 21 days was 128.7 ± 32.2. Reproduction at 16, 157 and 344 µg MOT(EHMA)/L was significantly reduced. Since reproduction at 19 and 36 µg MOT(EHMA)/L was not reduced, the NOEC was considered to be 36 µg MOT(EHMA)/L. At the test concentrations of 157 and 344 µg MOT(EHMA)/L, production of mobile young was significantly reduced.

Mean parental body length was not significantly reduced at concentrations up and including to 157 µg MOT(EHMA)/L. At the highest test concentration (344 µg MOT(EHMA)/L), body length was reduced by 12% compared to the control. This was considered a treatment related outcome.

Aborted eggs were observed in the control and all but the highest concentration tested. The incidence was not related to treatment. Immobile young were seen incidentally in the control and the lowest concentration. In both cases, only one parent produced 3 or 6 immobile young. These parents were observed to be in poor condition throughout the test and not included in calculations or statistical analysis. Further, significant numbers of immobile young were produced at 50% of the WAF (157 µg MOT(EHMA)/L).

Parental condition was not clearly related to treatment, with small and pale daphnias in all concentrations and the control. However, there was a dose dependent increase in the number of daphnia trapped at the surface, even though a test substance film or floating layer was not observed.

Test conditions for pH, temperature, dissolved oxygen, hardness and TOC remained within the limits prescribed by the protocol and the test was considered valid.

For the lethality endpoint, LC50 was calculated to be 228 µg MOT(EHMA)/L; the overall 21-day LOEC was 157 µg MOT(EHMA)/L and the overall NOEC was 36 µg MOT(EHMA)/L.

Concerns regarding the substances composition in particular the presence of the impurity EHTG mean that this study as a worst case scenario with regard to risk assessment but is not used for classification and labelling which is based on the newer and more reliable acute toxicity studies performed on a purer version of the substance. Further investigation of this end point is due to be performed.