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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:
key study
Study period:
15 Feb - 08 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Remarks:
LC-MS/MS and TOC
Details on sampling:
- Concentrations: Control, 10 mg/L (nominal)
- Sampling for determination of the test item: at least once within 7 d in fresh media at the start of an exposure interval and in old media at the end of an exposure interval (48 or 72 h). Furthermore once during the test, samples of the saturated solution and the control taken during the preparation phase of the saturated solution and during the exposure phase (i.e. at 24, 20, 0 and 48 h) were analyzed via LC-MS/MS and also via TOC-analysis to demonstrate the establishment of an equilibrium of the test item concentration.
- Sampling for the analytical monitoring: during the preparation of the saturated solution and at the start of an exposure interval, samples of the fresh media were taken at 24 h (after addition of the test item and a few minutes of stirring), at 20 h (approx. 4 h after the start of the stirring period during the preparation of the saturated solution) and at 0 h (after preparation of the saturated solution). At the end of an exposure interval (48 or 72 h), samples for analyses were taken from the test vessels. For analysis of the TOC, separate replicates of the saturated solution and the control were prepared without daphnids and food algae and analyzed. For the longest exposure interval of 72 h, samples were taken at the start (0 h) and at the end of the exposure interval (72 h) once within the test period.
- Sample storage conditions before analysis: all samples were stored at room temperature until sample preparation and in an autosampler until start of analysis, if necessary
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the solution was prepared with a nominal concentration of 10 mg/L in Elendt M4 medium one day prior to the start of each exposure interval. 22.48 µL test item were slowly added by pipette to 2 L Elendt M4 medium. The mixture was stirred slowly for 24 h with a magnetic stirrer at room temperature. Subsequently the solution was allowed to stand for 1 h for separation of the phases and then the saturated solution was removed from the center of the aqueous phase. Once during the test the establishment of an equilibrium of the test item concentration was shown by analysis during the preparation phase (0 and 4 h after stirring) and during the exposure phase (0 and 48 h).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: daphnids were bred at the test facility (origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany)
- Culturing conditions: in glass vessels (2 - 3 L) with approx. 1.8 L medium (Elendt M4), at approx. 20 °C, in an incubator, 16 h illumination; light intensity of max. 1500 Lux. Daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae with an algae cell density of > 10^6 cells/mL
- Age of daphnids at test start: < 24 h. Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within this period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: yes
- Food type and amount: Pseudokirchneriella subcapitata (1.06 - 1.40 mL) and Desmodesmus subspicatus (0.623 -0.786 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.
- Frequency: daily

ACCLIMATION
- Acclimation period: non necessary since culture medium same as test medium

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
158 - 172 mg CaCO3/L
Test temperature:
19.8 - 20.4 °C (water temperature)
pH:
7.06 - 8.45
Dissolved oxygen:
7.63 - 9.64 mg/L
Nominal and measured concentrations:
Nominal concentration: 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): beakers were loosely covered with watch glasses
- Fill volume: 50 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 10 (1 daphnid per replicate was used in the control)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 211
- Culture medium different from test medium: no
- Intervals of water quality measurement: once within 7 d, in fresh media at the start of an exposure interval (0 h) and in old media at the end of an exposure interval (48 or 72 h), in one replicate of the control and the saturated solution. The water quality parameters in fresh media were measured in an additional replicate without daphnids of the saturated solution and the control. At the end of an exposure interval, the water quality parameters of the old media were measured in a test vessel of the saturated solution and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: 1500 Lux

EFFECT PARAMETERS MEASURED: immobile daphnids were observed and recorded once per day and offspring was counted. Dead specimens were removed. The neonates were removed after counting and before addition of algae. The number of aborted eggs or dead offspring was recorded. Abnormalities (e.g. swimming behavior, number of males and winter eggs) were observed and recorded daily. At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of the saturated solution and the control were determined (these information were not used for determination of a NOEC). The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
- Range finding study: yes (static conditions, 48 h, 2 replicates)
- Test concentrations: control, 2 mg/L
- Results used to determine the conditions for the definitive study: no mortalities were observed in the range finding test
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Observations on body length and weight: the mean values of the body length (excluding the anal spine) of the parental daphnids in the saturated solutionwere 4.20 mm per daphnid and 4.50 mm per daphnid in the control group. The mean values of the dry weight of the parental daphnids were 0.80 mg per daphnid in thesaturated solution and 0.64 mg per daphnid in the control.
- Other biological observations: stillborn juveniles and aborted eggs produced by the parental daphnids after 21 days was 0. No males were observed in the control or in the test groups during the test. No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 2 mg/L (95% CI: 1.8 - 2.28 mg/L)
Reported statistics and error estimates:
Since no mortality appeared in this study, statistical evaluation for the adult mortality was not necessary. Further information on statistics can be found in the field any other information on results incl. tables.

ANALYTICAL RESULTS

The measured concentrations of the test item determined via LC-MS/MS during the preparation phase of the saturated solution and during the exposure were all below the LOQ (152 µg/L) of the analytical method. The measured TOC concentrations were around the LOQ of 2 mg C/L in all analyzed samples. This indicates that the test item is hardly soluble as well as completely hydrolyzed during the exposure. Since the test item concentrations could not be quantified, the nominal concentration of the test item was used for the evaluation of the NOEC and LOEC.

Table 1: Measured TOC Concentrations during the Definitive Test

Sampling date

Day 0

Fresh media

(0 h)

Day 2

Old media

(48 h)

Day 9

Fresh media

(0 h)

Day 12

Old media

(72 h)

Day 13

Preparation of the saturated solution
(0 h)

Day 13

Preparation of the saturated solution
(after 4 h stirring)

Day 14

Fresh media

(0 h)

Day 16

Old media

(48 h)

Nominal

test item

concentration

[mg/L]

Total Organic Carbon (TOC)

Measured concentration [mg C/L]

10.0

(saturated solution)

2.70

< 2.00

< 2.00

< 2.00

< 2.00

3.65

< 2.00

< 2.00

Control

2.66

< 2.00

3.02

< 2.00

< 2.00

< 2.00

< 2.00

< 2.00

BIOLOGICAL RESULTS

Table 2: Effects on Reproduction for all parental Daphnids

Nominal

test item concentration

Mean number of offspring per survived / introduced parental daphnid

[mg/L]

Mean

SD

CV

10.0

82.7

9.89

12.0

Control

93.7

13.6

14.5

Table 3: First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal

test item concentration

[mg/L]

Day of first appearance of living juveniles at the parental daphnid in replicate no.

First

appearance

1

2

3

4

5

6

7

8

9

10

mean day

10.0

(saturated solution)

8

8

8

9

8

9

9

9

8

8

8.4

Control

9

9

8

9

9

9

9

9

8

8

8.7

STATISTICAL ANALYSIS

No statistically significant difference of the reproductive output in comparison to the reproductive output in the control was determined at the saturated solution.

Adult mortality was not observed in this study. Therefore, no statistical evaluation was carried out for this parameter.

Number of living juveniles per survived parental Daphnid:

- Shapiro-Wilk's test on normal distribution: normality check was passed (p > 0.01).

- Levene's Test on variance homogeneity (with Residuals): the Levene test indicates variance homogeneity (p <= 0.010). Variance homogeneity check was passed (p > 0.01). Normal-distribution and variance-homogeneity requirements are fulfilled.

- Two-sample Welch-t-test Procedure (two sided test) with cumulative offspring per survived parent at 21 d: two-sample comparison of treatments with "Control". Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha (Control(c) and treatment(t) variance was applied: s^2(c)/nc + s^2(t)/nt, each).

Table 4: Two-sample Welch-t-test Procedure

Treatm. [mg/L] mean s df %MDD t t* sign.
control 93.7 13.58
10 82.7 9.89 16 12 -2.07 0.55

non-sign.

VALIDITY CRITERIA

Table 5: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality of the parent animals (femaleDaphnia) does not exceed 20% at the end of the test.

% mortality of the Adult Daphnids after 7, 14 and 21 days of exposure was 0.

 yes

The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93.7 in the control group

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables".
Conclusions:
The study conducted according to the OECD guideline 211 and GLP did not show any long term effects to the test organism Daphnia magna resulting in a NOEC (21 d) >= 10 mg/L (nominal).
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Observations on body length and weight: the mean values of the body length (excluding the anal spine) of the parental daphnids in the saturated solutionwere 4.20 mm per daphnid and 4.50 mm per daphnid in the control group. The mean values of the dry weight of the parental daphnids were 0.80 mg per daphnid in thesaturated solution and 0.64 mg per daphnid in the control.
- Other biological observations: stillborn juveniles and aborted eggs produced by the parental daphnids after 21 days was 0. No males were observed in the control or in the test groups during the test. No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: no

Description of key information

NOEC (21 d) ≥ 10 mg/L (nominal, OECD 211, Daphnia magna) based on read across from CAS 16415-12-6

Key value for chemical safety assessment

Additional information

Dimethoxymethyloctadecylsilane (CAS 70851-50-2) hydrolyses rapidly (2.6 h) to octadecylmethylsilanediol, which bears a low water solubility (3.6E-03 mg/L at 20-25°C). In accordance with Regulation (EC) No 1907/2006, Annex VII long-term testing should be considered for substances which are poorly soluble in water. Thus, long-term toxicity data on aquatic invertebrates were additionally taken into account for the environmental assessment. Since no data on the long-term toxicity for dimethoxymethyloctadecylsilane (CAS 70851-50-2) towards aquatic invertebrates were available, reliable data from the analogue substance hexadecyltrimethoxysilane (CAS 16415-12-6) were read across. Read across was performed according to Regulation (EC) No 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and the read across assessment framework (RAAF, ECHA 2017). Details on the read across justification can be found in the attached justification in the respective target entry and in the overall justification for grouping of substances attached in IUCLID Section 13.

The study with the read across substance hexadecyltrimethoxysilane (CAS 16415-12-6) was performed according to OECD 211 guideline and GLP (2017). Semi-static conditions were applied with a renewal rate of three times per week. D. magna was exposed to one single nominal concentration of 10 mg/L (saturated solution) and a control for 21 d. The solution was prepared by adding the appropriate amount of the test item into medium and stirring slowly for 24 h. After 1 h standing (for separation of the phases), the saturated solution was removed from the center of the aqueous phase. The substance is known to hydrolyse rapidly and therefore two analytical methods were applied in order to monitor the concentrations in the media – LC-MS/MS and TOC. TOC was determined during preparation of the saturated solution (stirring phase) at time points 0 and 4 h. Additionally the TOC was measured at the exposure time points of 0 h (fresh medium), 48 h and 72 h (both old medium). Hexadecyltrimethoxysilane was measured by LC-MS/MS in the fresh and old media as well as during the preparation time of the solutions. The measured concentrations of the test item determined via LC-MS/MS were in all cases below the LOQ (152 µg/L). The measured TOC concentrations on the other hand were around the LOQ of 2 mg C/L in all samples. This verifies that the substance is hardly soluble and that it was hydrolysed already during the preparation phase of the solution. Therefore it is concluded that the organisms were exposed to the hydrolysis product. Since the test item concentrations could not be quantified, the nominal concentrations were used for the expression of the effect values. The study did not indicate any toxic effects by the substance to the reproduction of Daphnia magna resulting in a NOEC of ≥ 10 mg/L.

Based on the results of the analogue substance, dimethoxymethyloctadecylsilane (CAS 70851-50-2) is not assumed to cause harmful long-term effects to aquatic invertebrates.