Reaction mass of decamethyltetrasiloxane; dodecamethylpentasiloxane; hexamethyldisiloxane; octamethyltrisiloxane

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-05-25 to 2002-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Solvent control, 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L

- Sampling method: Samples were taken weekly from the newly prepared (solvent) control and from three test substance concentrations. After 48 hours the same spent media were sampled again.

- Sample storage conditions before analysis: The samples taken were added by syringe through the septum to the sample bottles (glass vials, containing sodium chloride solution, hexane and a stirring bar).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Stock solutions were prepared at the start of the test and at each medium renewal time. The stock solution vessels were wrapped in aluminium foil until used. Appropriate amounts of test substance were weighed out in syringes and dissolved by injection into TBA (Tertiary butyl alcohol). Using a glass syringe, aliquots of 12.5 μL were used to dose the test substance through the septum of the cap of the test vessels.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L

- Dilution water: DSWL-E water prepared from ground water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM

- Source: TNO stock culture. Weekly cultures are initiated with 125 daphnids of the same age (circa 1 day) in approximately 4 litres of culture medium. The cultures are maintained at 20+/-1ºC under a 16 h light, 8 h dark regime with a 30 minute transition period. The cultures are discarded after 4 weeks.

- Feeding during test

- Amount: Each 125 mL test vessels received between 40 and 80x10E6 cells/mL of Chlorella sp and 1.5-3.0 mg of yeast. The food was added to the dilution water used to prepare the test media or via a syringe on days between medium renewals.

- Frequency: Daily


ACCLIMATION

- Acclimation conditions: same as test

- Type and amount of food: 4 x 10E9 algal cells (Chlorella sp.) and 0.13 g of yeast in 4 litres

- Feeding frequency: Daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

209 mg/L

Test temperature:

19.1-20.7ºC

pH:

7.5-8.0

Dissolved oxygen:

>1.8 mg/L (several low oxygen concentrations were measured in the spent concentrations, however based on the laboratory's opinion this did not affect the results of the study )

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal test substance concentrations: 0 (control), 0 (solvent control), 0.10, 0.18, 0.32, 0.56, 1.0, and 1.8 mg/L.

The two top doses were above the limit of water solubility to make sure that the test reached water solubility. 

- Over the 3-week test period the measured concentrations averaged 79.4% of nominal. Refer to Table 1 below.

Details on test conditions:

TEST SYSTEM

- Test vessel: Test was carried out in closed silanized glass vessels

- Type (delete if not applicable): closed

- Fill volume: 125 mL

- Aeration: none

- Renewal rate of test solution (frequency/flow rate): 3 times per week (Monday, Wednesday and Friday)

- No. of organisms per vessel: 1

- No. of vessels per concentration (replicates): 10

- No. of vessels per control (replicates): 10

- No. of vessels per vehicle control (replicates): 10

- Biomass loading rate: 1 daphnia per container
 
OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light-8 h dark


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Synthetic medium (DSWL-E)

- Total organic carbon: 1.91 mg/L

- Ca/mg ratio: 2:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: Temperature, pH, and dissolved oxygen were measured every 2-3 days in the spent media.


EFFECT PARAMETERS MEASURED: Mortalities and numbers of young produced were determined at each test medium renewal.


VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY

- Test concentrations: not reported

- Results used to determine the conditions for the definitive study: not reported

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: condition

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: condition

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.25-0.37

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The mean number of offspring in the control and solvent control was 130 and 106, respectively.  The mean number of offspring in the 0.10, 0.18, 0.32, and the 0.56 mg/l treatments was 88, 81, 66, and 22, respectively.  The 0.10 mg/l nominal (0.08 mg/l measured) results were not significantly different from controls so that was determined to be the NOEC for reproduction.  The reproduction LOEC was 0.18 mg/l nominal (0.14 mg/l measured). The 21-day LC50 was estimated to be 0.57 mg/l nominal (0.45 mg/l measured).  All animals died at 1.0 and 1.8 mg/l and 3 died at 0.56 mg/l nominal.   The condition of the organisms was evaluated qualitatively compared to the controls.  Effects on organism condition were observed at 0.56 mg/l, while no effects were observed at 0.32 mg/l or lower.  The NOEC for condition was 0.32 mg/l nominal (0.25 mg/l measured) and the LOEC was 0.56 mg/l nominal (0.44 mg/l measured).

Refer to Table 2 below for test results. Refer to Table 3 for calculation of the time weighted mean (carried out by the present reviewers, not present in original study report).

Reported statistics and error estimates:

The EC50 values and their confidence intervals were calculated using a maximum likelihood fitting procedure on a logistic model.

The LC50 values and their confidence intervals were calculated using a parametric model developed by Kooijman.

The NOEC and LOEC values were determined by comparing treatments with the Controls. Effects on mortality were assessed using a binomial test at the 95 and 99% significance level. Effects on reproduction were assessed using a Dunnett-test at the 95 and 99% significance level.

Table 2. Test results

Nominal test concentration (mg/L)	Survival at end of test (%)	Cumulative number of live young per female	Cumulative number of live young per female as percentage of Solvent Control
0 (Control)	90	130	123
0 (Solvent control)	100	106	-
0.10	100	88	83
0.18	100	81*	77
0.32	100	66*	63
0.56	70	22*	21
1.0	0	0*	0
1.8	0	0*	0

*Significantly different from Solvent Control (p<5%)

Some offspring were found dead in the controls and every concentration at one of the solution replacement times. 
Offspring were monitored for mortality on days 11, 13 and 18.  The mortality was ascribed to the pressure in the closed and completely filled test vessels (droplets noticed on the outside of some of the lid septums).

The mean measured concentration was 79.4% of control, so this number was used in the original study report to calculate measured concentrations.

Validity criteria fulfilled:

yes

Conclusions:

A 21-day EC50 value of 0.30 mg/L has been determined for the effects of the test substance on reproduction of Daphnia magna. A 21-day NOEC of 0.08 mg/L (arithmetic mean), equivalent to 0.085 mg/l (time-weighted mean, derived by reviewer) has been determined for the same endpoint.

Reference 2

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)

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: 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 pretest 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:

LOEC

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 analyzed 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 mean measured concentrations.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-08 to 2009-05-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Water samples were collected from alternating replicate test chambers of each treatment and control group three and four days prior to test initiation to confirm the operation of the diluter. 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 in the test system. All samples were collected mid-depth and placed directly into Teflon centrifuge tubes and processed immediately for analysis.

- Sample storage conditions before analysis: The samples were processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

Stock solutions were prepared three times during the test. For each preparation, a primary stock solution was prepared in DMF at a nominal concentration of 0.067 mg/ml. The primary stock solution was mixed by inversion and appeared clear and colourless. Proportional dilutions of the primary stock were made to prepare four additional secondary stock solutions at nominal concentrations of 0.0042, 0.0084, 0.017 and 0.034 mg/ml. The secondary stock solutions were inverted to mix, and appeared clear and colourless.

The five stock solutions were delivered to the diluter mixing chambers (at a rate of 155 μl/minute) where they were mixed with dilution water (at a rate of 155 ml/minute) to achieve the desired test concentrations of 0.42, 0.84, 1.7, 3 4 and 6.7 μg/l. The solvent control was prepared by injecting DMF into the mixing chamber assigned to the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.1 ml/l.

The test solutions in the test chambers for all treatments and control groups appeared clear and colourless at test initiation and termination. On Day 3, and until the end of the test, a clear surface slick was observed on the surface of the water in the 6.7 μg/l treatment group indicating that this test concentration was at or above the limit of water solubility.

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 the test laboratory.

- Culture water: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test.

- 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 two or 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 chamber was fed 0.75 ml of YCT and 1.5 ml of algae. While this amount of feed exceeds the OECD guideline( 2) 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 five adult daphnids used to supply neonates for the test were held for at least 17 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.

- Allocation of test organisms to treatments: 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 , plastic cups) until each chamber contained five daphnids. Each group of daphnids was then 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:

136-148 mg/L as CaCO3

Test temperature:

19.9-20.3°C

pH:

8.1-8.3

Dissolved oxygen:

≥5.4 mg/l

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Solvent control), 0.42, 0.84, 1.7, 3.4 and 6.7 µg/L.

Mean measured test concentrations in treated vessels: 0.016, 0.075, 1.11, 3.2 and 4.9 µg/L.

The results are interpreted with reference to mean measured concentrations.

Details on test conditions:

- Test apparatus: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (dimethylformamide) control, and a negative (dilution water) control. Syringe pumps were used to deliver the five test substance stock solutions and dimethylformamide (DMF) for the solvent control Into mixing chambers indiscriminately assigned to each treatment and the solvent control. The pumps w ere calibrated prior to the test. The stock solutions were diluted with well water !n 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 ln 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-litre Teflon-lined stainless steel aquaria filled with approximately 22 litres 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, approximately 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.

- Lighting: Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone®50) were used for illumination of the cultures and test chambers. A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at test initiation using a SPER Scientific Model 840006C light metre and was 174 lux over one representative test chamber.

- Temperature: The target test temperature was 20+/-1°C. Temperature was measured in each test chamber at test initiation and termination, and at weekly intervals during the test, using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation and weekly during the test using a liquid-in-hand glass thermometer.

- Dissolved oxygen and pH: 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. Measurements of pH were made in alternating replicate test chambers of each treatment and control group at test initiation and at weekly intervals thereafter. Dissolved oxygen was measured using a Thermo Orion 850A plus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion 525A plus pH meter.

- Hardness, alkalinity and specific conductance: Hardness, alkalinity and specific conductance were measured in alternate replicates of the negative (dilution water) control and the highest test concentration test at test initiation and at weekly intervals thereafter.

- Total organic carbon (TOC): TOC was measured in the dilution water at test initiation and termination.

- Biological Observations and Measurements: Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of dead and 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). Immobilty 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.:

>= 4.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 4.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 4.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: adult mortality and reproduction

Reported statistics and error estimates:

The data for all parameters passed the assumptions of normality and homogeneity. Analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (p = 0.05). Those treatments that were significantly different from the pooled or solvent control treatments were identified using Dunnett's t-test (p≤0.05). All statistical tests were performed using SAS (6) 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	<LOQ
0 (Solvent control)	<LOQ	<LOQ
0.42	0.0623	14.8
0.84	0.301	35.8
1.7	1.11	65.3
3.4	3.23	94.9
6.7	4.88	72.8

 

Table 2. Summary of test results

 

Mean measured concentration (μg/l)	Percentage survival at end of test	Mean number of young produced per reproductive day	Mean length (mm)	Mean dry weight (mg)
0 (Control)	95	9.0+/-0.83	5.2+/-0.050	1.1+/-0.16
0 (Solvent control)	100	4.6+/-0.73	5.1+/-0.050	0.99+/-0.057
0.016	95	5.0+/-0.22	5.1+/-0.096	1.05+/-0.017
0.075	100	3.9+/-1.08	5.2+/-0.18	0.83+/-0.042
1.1	90	5.9+/-1.38	5.2+/-0.58	0.99+/-0.084
3.2	100	4.2+/-0.40	5.1+/-0.050	1.01+/-0.041
4.9	90	5.2+/-0.50	5.2+/-0.10	1.09+/-0.12

*Statistically significant difference (p<0.05) compared with pooled control by Dunnett’s test

Validity criteria fulfilled:

yes

Conclusions:

A 21-day EC50 of >4.9 μg/l and NOEC of ≥4.9 μg/l have been determined for the effects of the test substance on adult mortality, reproduction and growth of Daphnia magna, based on measured values.

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-20 to 2010-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Water samples were collected from alternating replicate test chambers of each treatment and control group one and six days prior to test initiation to confirm the operation of the diluter. 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 in the test system. Additionally, stock and water samples were collected from test chambers two days after test termination due to questionable results at test termination. All samples were collected mid-depth and placed directly into 100 ml volumetric flasks and processed immediately for analysis.

- Sample storage conditions before analysis: The samples were processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

Stock solutions were prepared two times during the test. For each preparation, a primary stock solution was prepared in DMF at a nominal concentration of 10000 ng/ml. The primary stock solution was mixed by inversion and appeared clear and colourless. A second primary stock solution was prepared by proportional dilutions at a nominal concentration of 700 ng/ml. Proportional dilutions of the 700 ng/ml primary stock solution were then used to prepare the four additional secondary stock solutions at nominal concentrations of 44, 88, 180 and 350 ng/ml. The secondary stock solutions w ere inverted to mix, and appeared clear and colourless.

The five 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 4.4, 8.8, 18, 35 and 70 ng/l. The solvent control was prepared by injecting DMF into the mixing chamber assigned to the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.1 ml/l.

The test solutions in the test chambers for all treatments and control groups appeared clear and colourless at test initiation and termination.

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 the test laboratory.

- Culture water: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test.

- 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 Day 20 and then once on the last day of the test. At each feeding, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae. While this amount of feed exceeds the OECD guideline( 2) 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 four adult daphnids used to supply neonates for the test were held for at least 27 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 w ere produced during the holding period.

- Allocation of test organisms to treatments: At test initiation the juvenile daphnids w ere collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers (e g , plastic cups) until each chamber contained five daphnids. Each group of daphnids was then 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:

132-140 mg/l as CaCO3

Test temperature:

20.0-20.2°C

pH:

8.0-8.1

Dissolved oxygen:

≥5.5 mg/l

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Solvent control), 4.4, 8.8, 18, 35 and 70 ng/l.

Mean measured test concentrations in treated vessels: 3.6, 7.0, 16, 31 and 47 ng/l.

The results are interpreted with reference to mean measured concentrations.

Details on test conditions:

- Test apparatus: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (dimethylformamide) control, and a negative (dilution water) control. Syringe pumps were used to deliver the five test substance stock solutions and dimethylformamide (DMF) for the solvent control into 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-litre Teflon-lined stainless steel aquaria filled with approximately 22 litre 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, approximately 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.

- Lighting: Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone®50) were used for illumination of the cultures and test chambers. A photoperiod of l6 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at test initiation using a SPER Scientific Model 840006C light meter and was 236 lux over one representative test chamber.

- Temperature: The target test temperature was 20+/-1°C. Temperature was measured in each test chamber at test initiation and termination, and at weekly intervals during the test, using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation and weekly during the test using a liquid-in-hand glass thermometer.

- Dissolved oxygen and pH: 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. Measurements of pH were made in alternating replicate test chambers of each treatment and control group at test initiation and at weekly intervals thereafter. Dissolved oxygen was measured using a Thermo Orion 850A plus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion 525A plus pH meter.

- Hardness, alkalinity and specific conductance: Hardness, alkalinity and specific conductance were measured in alternate replicates of the negative (dilution water) control and the highest test concentration test at test initiation and at weekly intervals thereafter.

- Total organic carbon (TOC): TOC was measured in the dilution water at test initiation and termination.

- Biological Observations and Measurements: Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of dead and immobile daphnids w ere 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 w ere 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.:

>= 47 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and survival

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 47 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and survival

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 47 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: adult mortality and reproduction

Reported statistics and error estimates:

Analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (p = 0.05). Those treatments that were significantly different from the pooled or solvent control treatments were identified using Dunnett's t-test (p≤0.05). All statistical tests were performed using SAS (6) software.

There were <50% reductions in survival and reproduction at the end of the test and this precluded the calculation of EC50 values for these endpoints.

Table 1. Results of analysis of test media

 

Nominal concentration (ng/l)	Mean measured concentration (ng/L)	Mean measured concentration as percentage of nominal
0 (Control)	<LOQ	<LOQ
0 (Solvent control)	<LOQ	<LOQ
4.4	3.6	82
8.8	7.0	80
18	16	89
35	31	89
70	47	67

 

Table 2. Summary of test results

 

Mean measured concentration (ng/L)	Percentage survival at end of test	Mean number of young produced per reproductive day	Mean length (mm)	Mean dry weight (mg)
0 (Control)	90	9.2+/-0.53	5.1+/-0.058	1.09+/-0.14
0 (Solvent control)	95	9.9+/-1.49	5.2+/-0.058	1.12+/-0.066
3.6	95	11.5+/-0.30	5.1+/-0.000	1.04+/-0.058
7.0	90	10.5+/-0.34	5.1+/-0.050	1.05+/-0.052
16	100	11.7+/-1.42	5.1+/-0.058	1.00+/-0.058
31	100	11.0+/-1.42	5.1+/-0.058	0.96+/-0.039*
47	85	10.4+/-1.89	5.2+/-0.010	1.02+/-0.081

*Statistically significant difference (p<0.05) compared with pooled control by Dunnett’s test

Validity criteria fulfilled:

yes

Conclusions:

A 21-day EC50 of >47 ng/l and NOEC of ≥47 ng/l have been determined for the effects of the test substance on adult mortality, reproduction and growth of Daphnia magna. The results have been obtained under flow-through conditions and are expressed relative to mean measured concentrations of the substance.

Description of key information

HMDS: Long-term toxicity to invertebrates: 21-day EC₅₀ 0.30 mg/l and NOEC 0.08 mg/l (measured arithmetic mean concentration), reproduction of Daphnia magna.

L3: Long-term toxicity to invertebrates: 21-d NOEC ≥15 μg/l (measured, mean; highest concentration tested), survival and mobility of Daphnia magna.

L4: Long-term toxicity to invertebrates: 21-d NOEC: ≥4.9 μg/l (measured; highest concentration tested), adult mortality, reproduction and growth of Daphnia magna.

L5: 21-day NOEC: ≥47 ng/l (mean measured concentration) adult mortality, reproduction and growth of Daphnia magna.

Additional information

Constituent HMDS

A 21-d EC₅₀ value of 0.30 mg/l (measured, arithmetic mean) and NOEC value of 0.080 mg/l (measured, arithmetic mean, equivalent to 0.085 mg/l, time-weighted mean) have been determined for the effects of Constituent HMDS on reproduction of Daphnia magna (TNO, 2003). In view of the exposure regime it is likely that the test organisms were exposed primarily to the parent form of the tested substance, although some losses were observed based on measured concentrations in spent media.

 

Constituent L3

A 21-day EC₅₀ value of >15 μg/l (highest concentration tested) has been determined for the effects of Constituent L3 on survival and mobility of Daphnia magna. A NOEC of ≥15 μg/l has also been determined in the same test for effects on growth and reproduction (Wildlife International, 2010). In view of the use of flow-through test conditions it is likely that the test organisms were exposed primarily to the parent substance.

 

Constituent L4

A 21-day EC₅₀ of >4.9 μg/l and NOEC of ≥4.9 μg/l (measured, highest concentration tested) have been determined for the effects of Constituent L4 on adult mortality, reproduction and growth of Daphnia magna (Dow Corning, 2009). In view of the use of flow-through test conditions it is likely that the test organisms were exposed primarily to the parent substance.

 

Constituent L5

A 21-day EC₅₀ value of >47 ng/l and NOEC of ≥47 ng/l have been determined for the effects of Constituent L5 on adult mortality, reproduction and growth of Daphnia magna (Dow Corning, 2010). The results have been obtained under flow-through conditions and are expressed relative to mean measured concentrations of the substance. In view of the use of flow-through test conditions it is likely that the test organisms were exposed primarily to the parent substance.