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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:
2009-06-19 to 2010-01-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples were collected from alternating replicate test chambers of each treatment and control group two times prior to the start of the test to confirm the operation of the diluter. The results from the first set of pre-test samples were low so a second set was collected after an additional 4 days of equilibration. Only the results from the second set of samples were reported.

Water samples also were collected from alternating replicate test chambers at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to determine concentrations of the test substance.

All samples were collected at mid-depth, placed in teflon centrifuge tubes, and processed immediately for analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

Stock solutions were prepared four times during the test. For the first preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 340 μg a.i./mL. The stock solution was mixed by inversion, and appeared clear and colourless. Proportional dilutions of the primary stock were made to prepare four additional stock solutions at nominal concentrations of 21, 43, 85 and 170 μg a.i./mL. The stock solutions were delivered to the diluter mixing chambers (at a rate of 15.5 μL/minute) where they were mixed with dilution water (at a rate of 155 mL/minute) to achieve the desired test concentrations of 2.1, 4.3, 8.5, 17 and 34 μg a.i./L. For the three subsequent preparations, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 680 μg a.i./mL. The stock was mixed by inversion and appeared clear and colourless. Proportional dilutions of the primary stock were made to prepare four additional stock solutions at nominal concentrations of 43, 85, 170 and 340 μg a.i./mL. The stock solutions were delivered to the diluter mixing chambers (at a rate of 7.75 μL/min) where they were mixed with dilution water (at a rate of 155 mL/min) to achieve the desired concentrations of 2.1, 4.3, 8.5, 17 and 34 μg a.i./L. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (99.9%). The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber assigned to the solvent control. The concentration of DMF in the solvent control and all treatment groups during the in-life portion of the test was 0.05 mL/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland.

- Culture conditions: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.6 to 20.8ºC. The pH of the water ranged from 7.9 to 8.7. Dissolved oxygen ranged from 7.2 to 9.2 mg/L (≥80% of saturation).

- Feeding: During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. Daphnids were fed three times per day through Day 6 of the test and then were fed four
times per day until the last day of the test. At each feeding, each test compartment was fed 0.75 mL of YCT and 1.5 mL of algae. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.

- Test organisms: The three adult daphnids used to supply neonates for the test were held for at least 20 days prior to collection of the juveniles for testing, and had produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the
7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers (e.g., 10 mL glass beakers) until each chamber contained five daphnids. Each group of daphnids then was transferred to an indiscriminately assigned test compartment. All transfers were made below the water surface using wide-bore pipettes.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
136-140 mg/L as CaCO3
Test temperature:
Temperatures measured weekly in the test chambers ranged from 19.7 to 19.9°C, while the temperature measured continuously was approximately 20.0°C
pH:
8.1 to 8.3
Dissolved oxygen:
≥70% of saturation (6.3 mg/L)
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 0 (Control), 0 (Solvent control), 2.1, 4.3, 8.5, 17 and 34 μg a.i./L

- Mean measured concentrations in treated vessels: 1.9, 3.7, 6.8, 14 and 15 μg a.i./L

- The mean measured test concentrations represented 90, 86, 80, 82 and 44% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.
Details on test conditions:
- Apparatus: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus) were used to deliver the five test substance stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into the mixing chambers indiscriminately assigned to each treatment and the solvent control. The pumps were calibrated prior to the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and at weekly intervals thereafter. The flow of test water from each mixing chamber was split and allowed to flow into two replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at weekly intervals thereafter to ensure that flow rates varied by no more than ±10% of the mean for the two replicates. The diluter flow rate was adjusted to provide approximately five volume additions of test water in each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test.

- Test chambers: Test chambers were 25-L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test solution. The daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 mL glass beakers, 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative test compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. The test chambers were placed in a temperature-controlled water bath to maintain the target temperature throughout the test period. All test chambers were labelled with the project number, test concentration and replicate.

- Dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water.

- Lighting: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.

- Temperature: Temperature was measured in each test chamber at test initiation and termination, and at weekly intervals during the test. Temperature also was monitored continuously in the Replicate A test chamber of the negative control.

- Dissolved oxygen: Dissolved oxygen was measured in alternating replicate test chambers of each treatment and control group at test initiation and termination, and approximately three times per week during the test.

- Biological observations: Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 15 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, growth and reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 15 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: adult survival and mobility
Reported statistics and error estimates:
Test endpoints analysed statistically for first-generation daphnids were survival, reproduction, and growth (length and dry weight). EC50 values were determined based on reproduction and on the immobility observed in the first-generation daphnids at the end of the test. The NOEC was defined as the highest test concentration that produced no significant treatment-related effects on survival, reproduction or growth. The LOEC was defined as the lowest test concentration that produced a significant treatment-related effect on survival, reproduction or growth. The MATC was calculated as the geometric mean of the NOEC and LOEC. All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

Table 1. Results of analysis of test media 

Nominal concentration (μg/L)

Mean measured concentration (μg/L)

Mean measured concentration as percentage of nominal

0 (Control)

<LoQ

-

0 (Solvent control)

<LoQ

-

2.1

1.9

90

4.3

3.7

86

8.5

6.8

80

17

14

82

34

15

44

 

Table 2. Test results 

Nominal concentration (μg/L)

Percent Adult

Survival

Mean Number Young

Per Reproductive Day

± Std. Dev.

Mean Length

± Std. Dev.

(mm)

Mean Dry Weight

± Std. Dev.

(mg)

0 (Control)

90

11.8 ± 0.77

5.3 ± 0.12

0.94 ± 0.086

0 (Solvent control)

100

11.5 ± 1.28

5.3 ± 0.096

1.03 ± 0.14

0 (Pooled control)

95

11.6 ± 0.99

5.3 ± 0.099

0.98 ± 0.12

2.1

100

11.8 ± 0.38

5.4 ± 0.058

1.06 ± 0.038

4.3

90

12.9 ± 1.36

5.2 ± 0.096

1.03 ± 0.19

8.5

90

10.2 ± 2.13

5.2 ± 0.15

1.02 ± 0.035

17

100

11.2 ± 2.64

5.4 ± 0.10

1.03 ± 0.13

34

90

11.5 ± 0.66

5.2 ± 0.096

0.98 ± 0.021

There were no statistically significant decreases in comparison to the pooled control using Dunnett’s test (p≤ 0.05).

Validity criteria fulfilled:
yes
Conclusions:
A 21-day EC50 of >15 μg/L has been determined for the effects of the test substance on survival and mobility of Daphnia magna . A NOEC of ≥15 μg/L has been determined in the same test for effects on growth and reproduction. The results were obtained under flow-through test conditions and are expressed as arithmetic mean measured concentrations.
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:
See endpoint summary for justification of read-across
Reason / purpose:
read-across source
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 15 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, growth and reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 15 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: adult survival and mobility

Description of key information

Long-term toxicity to invertebrates: 21-d NOEC ≥0.015 mg/l (mean measured concentration) (highest concentration tested) (OECD Guideline 211 (Daphnia magna Reproduction Test)), read-across from an analogous/structurally related substance octamethyltrisiloxane (CAS 107-51-7).

Key value for chemical safety assessment

Additional information

There are no reliable long-term invertebrate toxicity data available for 1,1,1,3,5,5,5 -heptamethyltrisiloxane (CAS 1873-88-7), therefore good quality data for an appropriate structural analogue, octamethyltrisiloxane (CAS 107-51-7), have been read across.

A 21-day EC50 value of >0.015 mg/l (arithmetic mean measured concentration) (highest concentration tested) have been determined for the effects of octamethyltrisiloxane (CAS 107-51-7) on survival and mobility of Daphnia magna. A NOEC of ≥0.015 mg/l has also been determined in the same test for effects on survival, growth and reproduction.

In view of the exposure regime it is likely that the test organisms were exposed predominantly to the tested substance. Refer to the discussion in the Section 6 endpoint summary (Section 7.0 of CSR) for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.