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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:
18 Mar 2013 to 25 Apr 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
03 October 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard E1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests (2004)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Analytical purity: 97.41 %
- Lot/batch No.: A022C7C003
- Expiration date of the lot/batch: July 12, 2013
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Each treatment and control group
- Sampling method: Samples were collected from each treatment and control group approximately 13 days prior to the start of the test after conditioning the diluter for approximately two days. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. At test termination, additional backup samples were collected for possible analysis if needed. All samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.
- Time schedule: Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at test initiation, at approximately weekly intervals during the test and at test termination.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: vehicle control
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.02 mL/L
- Method: Individual stock solutions were prepared for each of the five concentrations tested, and were prepared twice during the study. At each preparation, a primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 200 mg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 12.5, 25, 50 and 100 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and were all clear and colorless. Stock solutions were stored refrigerated in glass amber bottles or glass graduated cylinders, and aliquots of each stock were placed in the syringe every 3 or 4 days during the study. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all hydroxyethyl acrylate treatment groups was 0.02 mL/L.
- Preparation: Delivery of the test substance to the test chambers was initiated 15 days prior to the introduction of the daphnids to the test water in order to achieve equilibrium of the test substance in the test chambers.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: waterflea
- Source: Wildlife International, Easton, Maryland
- Age of test stock: Neonates, < 24 hours

ACCLIMATION
The seven adult daphnids used to supply neonates for the test were held for 21 days prior to collection of the juveniles for testing, and had each 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. To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. 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
Post exposure observation period:
not applicable
Hardness:
128 to 140 (mg/L as CaCO3)
Test temperature:
20 ± 1°C
pH:
8.0 to 8.1
Dissolved oxygen:
≥ 6.2 mg/L (≥ 69% of saturation)
Salinity:
no data
Conductivity:
360-385 µS/cm
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0 mg/L (based on a range-finding study)
- Measured concentrations: -, 0.21, 0.41, 0.86, 1.8, 3.8 mg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 25-L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test water. 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 compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm.
- Type of flow-through: Syringe pumps were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers.
- Renewal rate: The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 3.1 μL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations. The negative control received dilution water only.
- No. of organisms per vessel: 10 (Each replicate contained two compartments with five daphnids)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Feeding during test: yes
- Food type: mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Amount: Each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae and 0.5 mL of vitamin solution. This amount of feed is equal to approximately 0.7 mg C/daphnid/day. 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.
- Frequency: three times per day through Day 7 of the test and then four times per day until Day 20 and once at the last day of the test

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: <1 mg C/L
- Metals: according to OECD guideline
- Pesticides: according to OECD guideline
- Alkalinity: 178 to 182 mg/L as Ca CO3
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 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.
- Light intensity: 428 lux

EFFECT PARAMETERS MEASURED: growth, immobilisaton and reproduction.
- First-generation daphnids were observed daily during the test for mortality, the onset of reproduction, and clinical signs of toxicity (inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week (i.e., Monday, Wednesday and Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 3.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
length and dry weight
Details on results:
See 'Any other information on results incl. tables'.
- Survival of parent animals: Survival in the pooled controls, 0.21, 0.41, 0.86, 1.8 and 3.8 mg/L treatment groups at test termination was 93, 95, 100, 95, 95 and 100%, respectively.
- No. of offspring produced per day per female: Adult daphnids in the pooled controls, 0.21, 0.41, 0.86, 1.8 and 3.8 mg/L treatment groups produced an average of 10.1, 9.3, 9.6, 10.2, 9.7 and 7.9 live young per reproductive day, respectively. There was a statistically significant decrease in mean neonate production in the 3.8 mg/L treatment group in comparison to the pooled controls.
- Body length and weight of parent animals: Daphnids in the pooled controls, 0.21, 0.41, 0.86, 1.8 and 3.8 mg/L treatment groups had mean lengths of 4.4, 4.4, 4.4, 4.4, 4.2 and 4.0 mm, respectively, and mean dry weights of 0.90, 0.95, 0.84, 0.83, 0.71 and 0.59 mg, respectively.
- Time to first brood release: 8 days
- Other biological observations: Daphnids in the 0.21, 0.41, 0.86, 1.8 and 3.8 mg/L treatment groups that survived to test termination generally appeared normal.
- Effect concentrations exceeding solubility of substance in test medium: The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Test endpoints analyzed statistically for first-generation daphnids were survival, reproduction, and growth (length and dry weight). Reproduction was based on the number of live young produced per reproductive day. Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.
Survival data was considered to be discrete-variable data, while reproduction and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the control means were identified using Dunnett’s test (α = 0.05). EC50 values were determined based on reproduction and on the immobility observed in the first-generation daphnids at the end of the test. All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

Table: Summary of Survival, Reproduction and Growth

Mean measured concentration (mg/L)

Percent Adult Survival

Mean No. Neonates per reproductive day ± SD

Mean Length ± SD (mm)

Mean Dry Weight ± SD (mg)

Negative Control

90

10.1 ± 0.53

4.4 ± 0.13

0.89 ± 0.09

Solvent Control

95

10.1 ± 0.53

4.4 ± 0.08

0.90 ± 0.10

Pooled control

93

10.1 ± 0.49

4.4 ± 0.10

0.90 ± 0.09

 0.21

95 #

9.3 ± 0.56

4.4 ± 0.10

0.95 ± 0.11

 0.41

100

9.6 ± 0.99

4.4 ± 0.05

0.84 ± 0.11

 0.86

95

10.1 ± 1.7

4.4 ± 0.10

0.83 ± 0.12

 1.8

95

9.7 ± 1.1

4.2 ± 0.10 *

0.71 ± 0.04 *

 3.8

100

7.9 ± 1.3 *

4.0 ± 0.15 *

0.59 ± 0.10 *

* Indicates statistically significant differences in growth and reproduction from the pooled controls (Dunnett’s one-tailed test, p≤0.05).

# One daphnid was accidentally killed during transfer on Day 10 of the test. Therefore, it was excluded from the analysis of the survival end point.

 

ADDITIONAL EFFECT VALUES

- Survival (based on immobility): EC50 > 3.8 mg/L

- Reproduction: LOEC: 3.8 mg/L

- Growth (length and dry weight): LOEC: 1.8 mg/L

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables' (validity criteria were manually checked by the assessor).
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
GLP-guideline study performed by japanese authorities, no study report available for review.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in report): 2-hydroxylethyl acrylate
- Analytical purity: no data
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Daphnia magna
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.48 mg/L
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.74 mg/L
Conc. based on:
test mat.
Basis for effect:
reproduction

Description of key information

The 21-d NOEC value (based on growth) is 0.86 mg/L in aquatic invertebrates (Daphnia magna).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.86 mg/L

Additional information

Two studies are available that assessed the acute toxicity of 2-hydroxyethyl acrylate to aquatic invertebrates.

The long term toxicity of 2-hydroxyethyl acrylate to aquatic invertebrates was determined in a study conducted by Wildlife International according to OECD TG 211 (Wildlife International, 2013). The cladoceran, Daphnia magna, was exposed to hydroxyethyl acrylate at mean measured concentrations of 0.21 to 3.8 mg/L under flow-through conditions for 21 days. There were no statistically significant treatment-related effects on survival at concentrations ≤3.8 mg/L. A statistically significant treatment-related effect on reproduction was noted at the 3.8 mg/L treatment concentration. Growth, measured as length and dry weight, was the most sensitive biological endpoint measured in this study. Daphnids exposed to hydroxyethyl acrylate at concentrations ≥1.8 mg/L had statistically significant reductions in length and weight in comparison to the pooled controls. Consequently, the NOEC, based on growth, was 0.86 mg/L, the LOEC was 1.8 mg/L and the maximum acceptable toxicant concentration (MATC) was calculated to be 1.2 mg/L. The 21-day EC50 values for adult immobility and reproduction were greater than 3.8 mg/L, the highest concentration tested.

In another study 21-day chronic reproduction test on Daphnia magna in accordance with OECD guideline 211 the NOEC was determined to be 0.48 mg/L (MOE, 1988). Although no detailed test report is available, all tests from the Japanese Authorities are performed according to OECD guidelines and GLP.

Overall, the effect value of 48-h EC50 value of 0.86 mg/L was selected as key value for the chemical safety assessment, as it is considered the most reliable result (derived in a GLP-compliant study that followed standard guidelines, the daphnids in this study were exposed under flow-through condtitions and the effect value is based on measured concentrations).