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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-12-01 to 2010-01-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study with restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The concentrations of the test item SILRES® BS 1701 were measured in all duplicate test media samples from the dilution 1:3.2 and from the undiluted filtrate, determined in the experiment as the 21-day NOELR and LOELR. From the control samples, one sample per sampling date was analyzed.

For determination of the test item concentrations, duplicate samples were taken from the freshly prepared test media of all test concentrations and from the control at one treatment period of the first, second and last week (Day 0, 7 and 16, respectively).

To determine the maintenance of the test item concentrations in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).

Immediately after the samples were taken, an appropriate amount of methanol (90 mL) was added to each sample to stabilize the test item during the storage period. Then the samples were stored deep-frozen (at about –20 °C) till the date of analysis.
Vehicle:
no
Details on test solutions:
Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 130.0 mg of test item in 1300 mL of test water or 200 mg of test item (effective weights: 199.4-200.3 mg) in 2000 mL of test water. The dispersion was supported by ultrasonic treatment for 15 minutes and intense stirring by a magnetic stirrer over 96 hours in the dark, to dissolve a maximum amount of the test item in the dispersion.

The long stirring period of 96 hours was selected according to the results of a pre-experiment (non-GLP) which showed that the maximum concentration of test item was reached after the stirring period of 96 hours.

After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The negative pressure of the filtration unit was reduced as far as possible. The test water (containing the water dissolvable part of the test item) was taken from the middle of the water column to avoid any undissolved test item in the test water. This undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= addition of daphnids).
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organisms: Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test.

Culture conditions: The temperature and light conditions were identical to those of the test.

Feeding during culture: During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen,
37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).

Culture vessels: Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The animals were fed normally three times a week with green algae of the species Scenedesmus subspicatus and with a
fish food suspension.

Observations: The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.

Test organisms: The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Hardness: 2.5 mmol/L (250 mg/L as CaCO3)

Alkalinity: 0.9 mmol/L
Test temperature:
20 to 22 °C
pH:
7.4 to 8.2
Dissolved oxygen:
≥7.7 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal treatments: 100 mg/L WAF and dilutions of 1:3.2, 1:10, 1:32 and 1:100 were used as test media.

The mean measured test item concentrations in the test media of the undiluted filtrate (loading rate 100 mg/L) and the 1:3.2 dilution were 376 and 58 μg/L, respectively.

It is likely that the test medium also contained hydrolysis products of the substance but these were not quantified.
Details on test conditions:
Test design: The study was started with 10 daphnids per treatment. Each test animal was kept individually in a glass beaker. The test animals were randomly distributed to the test vessels. The test duration was 21 days.

Test conditions: This semi-static test with test medium renewals every 48 or 72 hours was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20-22 °C. A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 520 and 680 Lux.

Test medium renewal: The test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium.

Feeding of test organisms: The test animals were fed daily (with the exception of Day 3, when daphnids were not fed) with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension. The fish food suspension was fed in addition to the algal food, because a toxic effect of the test item on the algae could not be excluded and to reduce the amount of algal food to prevent an increase of the pH values in the test media due to algal growth. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.

Observations: The test replicates were observed for mortality of adults on Days 0-2 and thereafter at least three times per week before renewal of the test media. On the same dates, the test replicates were observed for live and dead offspring and for the presence of aborted eggs.

Monitoring of test conditions: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and of the control. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored.

Test medium observations: The appearance of the test media was visually inspected and recorded at the beginning and end of each test medium renewal period.

Range-finding test: The selection of the test concentrations was based on the results of a range-finding test (non-GLP).
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
58 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
376 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate but based on hydrolysis half-life exposure is likely to be mainly to hydrolysis product.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate but based on hydrolysis half-life exposure is likely to be mainly to hydrolysis product.
Basis for effect:
reproduction
Reported statistics and error estimates:
The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The mean reproduction rates of the daphnids at the test concentrations were compared to the control by multiple Williams’ tests.

The EC50 of the reproduction rate after 21 days could not be calculated due to the low toxic effect of the test item on the reproduction rate up to the highest test concentration.

Table 1. Test results

Treatment

Mean measured test substance concentration (μg/L)

Percentage adult survival at end of test

Mean (+/-SD) number of live offspring per surviving adult at end of test

Control

<10.1 (LOQ)

90

95.0 (+/-14.2)

1:100 WAF dilution

Not determined

100

92.9 (+/-8.7)

1:32 WAF dilution

Not determined

80

98.6 (+/-11.5)

1:10 WAF dilution

Not determined

100

97.7 (+/-17.5)

1:3.2 WAF dilution

58

90

92.8 (+/-32.4)

Undiluted 100 mg/L WAF

376

90

73.7 (+/-21.9)*

*Significantly different from Control

It is inappropriate to interpret the results of the test with reference to the dilution ratios used in the preparation of the test media.

The test results have therefore been interpreted with reference to mean measured concentrations of the test substance. It is likely that the test medium also contained hydrolysis products of the substance but these were not quantified.

Validity criteria fulfilled:
yes
Conclusions:
A 21-day NOELR of 32 mg/L and LOELR of 100 mg/L have been determined for the effects of the test substance on reproduction of Daphnia magna. Loading rates above 0.1 mg/L are above the water solubility of the substance (<0.1 mg/L). The concentration of test substance in the test medium corresponding to the NOELR was 58 μg/L based on mean measured concentrations over the period of the test. It is therefore likely that the test medium contained predominantly hydrolysis products of the substance but these were not quantified. It is also possible that the presence of undissolved material may have contributed to the effects observed on reproduction in the undiluted dispersion however there is no conclusive evidence for this.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-04-07 to 2009-04-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples were taken from the freshly prepared test media of the loading rate of 100 mg/L and the control at the start of every test interval. In addition, duplicate samples were taken from the freshly prepared (Days 0, 2, 5 and 7) and aged test media (Days 2, 5 and 7) of all test concentrations and from the control at three test intervals. The aged samples were stored without food and test animals, but were incubated during the renewal periods under test conditions.

- Sample storage conditions before analysis: All samples were analyzed immediately after the sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A dispersion of the test item with the loading rate of 100 mg/L was prepared by adding 300 mg of the test item (dosed by pipetting 300 μL of the test item) into a beaker partially filled with test water under intense stirring. No auxiliary solvent or emulsifier was used. After addition of the test item, the beaker was filled up with test water to the volume of 3000 mL and the test item was mixed into the test water as homogeneously as possible using ultrasonic treatment (15 minutes). The pH was adjusted from 3.0 to 7.9 (± 0.3). Thereafter, the dispersion was stirred on a magnetic stirrer at room temperature over 24 hours in the dark. The stirring period of the dispersion was chosen according to the results of a pre-experiment.

Following the stirring period of 24 hours, the test medium was incubated in the dark for an equilibration period of one hour to allow any undissolved reaction products to float at the surface of the test solution or to settle on the bottom. The test water (containing the water dissolvable hydrolysis products) was taken from the middle of the water column to avoid any undissolved test item/hydrolysis products in the test water.

This test water was used as the highest concentrated test medium and as the stock solution for lower concentrated test media. Requisite aliquots were diluted with test water to obtain the additional test concentrations.

The test media were freshly prepared before the start of the test and before each test medium renewal.

- Controls: Dilution water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Strain/clone: Clone 5

- Source: University of Sheffield/UK

- Age of parental stock (mean and range, SD): The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.

- Feeding during test: The test animals were fed daily with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension.

The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.

The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.


ACCLIMATION

- Acclimation conditions (same as test or not): The temperature and light conditions were identical to those of the test.

- Feeding frequency: The animals were fed normally three times a week with green algae of the species Scenedesmus subspicatus and with a fish food suspension.

- Health during acclimation (any mortality observed): No signs of stress were observed and the brood stock was healthy.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
250 mg/L as CaCO3
Test temperature:
20-21°C
pH:
7.5-8.0
Dissolved oxygen:
≥7.2 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 100 mg/L and dilutions of 1:2.16, 1:4.7, 1:10 and 1:22.

Mean initial measured concentrations in the treated media: 65, 32, 14, 6.7 and 3.1 mg/L

At the start of the renewal periods the concentrations of the test item measured in the test medium of all tested concentrations (based on the TOC) were between 41 and 76% of the nominal concentrations. At the end of the three measured renewal periods the concentrations were between 62 and 85% (test media without food). Thus, the TOC content of the tested concentrations was sufficiently stable during the test medium renewal periods of two and three days. All reported biological results are related to the initially mean measured concentrations of the test item.
Details on test conditions:
TEST SYSTEM

- Test vessel: The test was performed in 100-mL glass beakers containing 80 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.

- Replication: The study was started with 10 daphnids per treatment. Each test animal was kept individually in a glass beaker. The test animals were randomly distributed to the test vessels. The test duration was 21 days.

- Test medium renewal: The test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes into test vessels containing freshly prepared test medium.

- Lighting: A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 520 and 680 Lux.

- Observations: The test replicates were observed for mortality of adults on Days 0-2 and thereafter three times per week before renewal of the test media. On the same dates, the test replicates were observed for live and dead offspring and for the presence of aborted eggs.

- Calculations: The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.

- Water quality: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and of the control. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored. The appearance of the test media was visually inspected and recorded at the beginning and end of each test medium renewal period.

- Selection of test concentrations: test concentrations were selected on the basis of the results of a non-GLP range-finding test (results not reported)
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Mean initial measured concentration of Total Organic Carbon expressed as equivalent test substance concentration
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
65 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Mean initial measured concentration of Total Organic Carbon expressed as equivalent test substance concentration
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Control mortality: 0%

- Other biological observations: With the exception of the reported mortality, no visible abnormalities were observed at the test animals during the test. No EC values for the inhibition of the reproduction rate could be calculated since no effect was determined on the reproduction of the daphnids at all test concentrations with surviving parent animals.
Reported statistics and error estimates:
The mean reproduction rates of the daphnids at the test concentrations were compared to the control by multiple Williams’ tests

Table 1. Results of analysis of test media

 Nominal treatment  Nominal concentration (mg/L)  Mean measured initial concentration (mg/L)  Percentage of nominal  n
 0 (Control) 0  -  -  -
 1:22 dilution of stock  4.5  3.1  68  4
 1:10 dilution of stock  10 6.7   67  4
 1:4.7 dilution of stock  21  14  68  4
 1:2.16 dilution of stock  46  32  69  4
 100 mg/L stock  100  65  65  9

Table 2. Test results

 Mean initial measured concentration (mg/L)  Percentage mortality after 21 days  Day of first offspring release  Mean reproduction rate (live young per surviving adult) Mean reproduction rate as a percentage of control 
 0 (Control)  0  9  89.8  -
 3.1  20  7  86.1  96
 6.7  10  7  92.4  103
 14  10  9  84.3  94
 32  20  9  82.5  92
 65  100  7  Not applicable (all parents dead)
Validity criteria fulfilled:
yes
Conclusions:
A 21-day NOEC of 32 mg/L and a LOEC of 65 mg/L have been determined for the effects of the test substance on mortality of Daphnia magna. No effects on reproduction were determined in test concentrations below the LOEC for mortality. The report authors note that it cannot be excluded that the effects of the test item were at least partly caused by undissolved test item, which could not be removed by the preparation method used during the
test period. It is likely that the test organism were primarily exposed to the hydrolysis products of the substance.

Description of key information

A 21-day NOEC of 32 mg/l has been determined for the effects of read-across substance trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4) on mortality of Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
32 mg/L

Additional information

A 21-day No Observable Effect Loading Rate (NOELR) of 32 mg/l and Lowest Observable Effect Loading Rate (LOELR) of 100 mg/l have been determined for the effects of the test substance on reproduction of Daphnia magna (Harlan, 2010a). A NOELR value result has been used in this instance because the loading rate of the test substance in the test solution was well above the test substance solubility. The NOELR refers to the point at which no effects were observed in relation to the amount of test substance put into the test solution, rather than the actual concentration of the test substance in the test solution, where a NOEC value would be used instead. A NOEC of 0.058 mg/l was defined on the basis of measurements of the parent substance in the no effect concentration. However it is not appropriate to use this value for risk assessment purposes as (i) the concentration of the equally relevant hydrolysis products were not considered by the analytical method and did therefore not contribute to the measured value of the no effect concentration, (ii) the effect concentration was clearly above the water solubility limit (<0.1 mg/l) of the parent substance in terms of nominal (100 mg/l) and measured (376 µg/l) values and it is not expected that the undissolved substance could easily be hydrolysed or be removed by a filtration processes (see discussion in short-term toxicity to invertebrates data). Based on the available data it is therefore not possible to (i) quantify a reasonable no effect concentration (NOEC) for the silane and its hydrolysis products and (ii) to separate physical from systemic effects at the effect concentration.

 

Instead, a long-term study with Daphnia magna has been read across from trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4) because its silanol hydrolysis product is the same as that of the registration substance. Effects were seen in the highest concentration only. Due to the high reactivity of the chlorosilane (hydrolysis half-life <1 minute at pH 7), the no effect concentration was determined by DOC analysis (which would measure both parent and hydrolysis product). This resulted in a measured NOEC of 32 mg/l (Harlan, 2009). The test was carried out in accordance with OECD TG 211, and in compliance with GLP. The test organisms would have been exposed to the hydrolysis products of the substance, (2,4,4-trimethylpentyl)silanetriol and hydrochloric acid.

 

While the data are considered to be sufficient to derive a reliable hazard assessment for the silanol hydrolysis product of the registered substance, an OECD TG 211 chronic Daphnia reproduction test is being conducted with the registration substance according to ECHA final decision TPE-D-2114455990-41-01/F. The test is currently ongoing. The substance will be updated once results of the study are available and the study is finalised. An update is planned to be completed within three months from when the last final report is received. The draft report is expected in January/February 2021, with the final report following in March/April 2021.

Refer to the discussion in the IUCLID Section 6 endpoint summary or Section 7.0 of the CSR for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.