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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13/08/2012-25/01/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP comparable to OECD guideline
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Qualifier:
according to
Guideline:
other: ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests
Principles of method if other than guideline:
/
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
/
Analytical monitoring:
yes
Details on sampling:
- Concentrations: samples were collected from all concentrations (negative control, solvent control, 1.9; 3.8; 7.5; 15; 30 µg/L)
- Sampling method:All samples were collected from mid-depth, placed in separatory funnels
- Sample storage conditions before analysis: Analyzed immediately.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Individual stock solutions were prepared for each of the five concentrations tested, and were prepared twice during the study. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 1500 µg/mL. The primary stock solution was sonicated for 30 minutes, followed by inversion to mix. Four secondary stock solutions were prepared in DMF at nominal concentrations of 95, 190, 375 and 750 µg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by inversion.
- Controls:negative control (dilution water only), solvent control (was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.02mL/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Wildlife International, Easton, Maryland.
- Feeding during test: a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Amount: At each feeding, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae.
- Frequency: Daphnids were fed two or three times per day through Day 7 of the test and then were fed four times per day until the last day of the test.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: same as test
- Feeding frequency: same as test
- Health during acclimation (any mortality observed): The adults showed no signs of disease or stress and no ephippia were produced during the holding period.



METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: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. 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:
/
Hardness:
139 +/- 5 mg/L CaCO3
Test temperature:
20 +/- 1°C
pH:
8.1 - 8.2
Dissolved oxygen:
>= 60% of saturation (5.5mg/L)
Salinity:
/
Nominal and measured concentrations:
Nominal concentrations: 1.9; 3.8; 7.5; 15; 30 µg/L
Measured concentrations: 1.9; 3.9; 5.7; 13; 23 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test water
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) 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.
- Renewal rate of test solution (frequency/flow rate): 5 volume additions of test water in each test chamber per day.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: obtained from a well approximately 40 meters deep located on the Wildlife International site.
- Total organic carbon: < 1 mg C/L
- Particulate matter:
- Metals: see Appendix 4
- Pesticides: see Appendix 4
- Chlorine: /
- Alkalinity: 178 mg/L as CaCO3
- Ca/mg ratio: 2.64
- Conductivity: 335 µS/cm
- Salinity:/
- Culture medium different from test medium: No
- Intervals of water quality measurement:preceding the test, day 7, day 14, day 21

OTHER TEST CONDITIONS
- Adjustment of pH:/
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity:333 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival, reproduction, growth

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
23 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth, survival and reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 23 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, growth and reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 23 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Details on results:
- Mortality of parent animals: 0% for all concentrations, except 3.9 µg/L: 10%
- No. of offspring produced per day per female: 10.22
- Body length and weight of parent animals: 4.75 mm and 0.76
- Type and number of morphological abnormalities: /
- Type and number of behavioural abnormalities:/
- Number of males and females (parental): /
- Time to first brood release or time to hatch: Day 8
- Brood size: Adult daphnids produced an average of 9.4, 10.8, 10.0, 10.8 and 10.1 in the 1.9, 3.8, 5.7, 13 and 23 µg/L treatment groups.

Results with reference substance (positive control):
/
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.
Negative control and solvent control data for each parameter were compared using an appropriate statistical test. No significant differences between the control groups were found (α = 0.05) in the survival, length or dry weight data. Therefore, the control data for those parameters were pooled for comparison with the treatment groups. Since the negative and solvent control reproduction data were significantly different (α = 0.05), the reproduction data from the treatment groups were compared to both the negative control and solvent control data separately
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 pooled controls. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s or Chi-square tests 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 one-tailed test (α = 0.05). All statistical tests were performed using a personal computer with SAS (6) or TOXSTAT (9) software.
Validity criteria fulfilled:
yes
Conclusions:
scientific judgement was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. 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.
In this study the highest reported water solubility value was lower than the actual value reported in the water solubility study. This difference in solubility values is explained by the hydrophobicity property of the substance and the difference between the experimental media (reagent water vs media with organisms). There is a decrease in concentrations of the test substance along the experiment.
Executive summary:

Title: Brominated Epoxy Having Epxoy Equivalent of 400 gr/eq: A Flow-Through Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna)

Length of exposure: 21 days

Test organism: Cladoceran (Daphnia magna)

Source of test organism: Wildlife International , Easton, Marylan 21601

Test concentrations: Nominal: negative, solvent control, 1.9, 3.8, 7.5, 15, 30 µg/L.

Mean measured: < LOQ, < LOQ, 1.9, 3.9, 5.7, 13, 23 µg/L

Results: Based on mean test concentrations:

No-Observed-Effect-Concentration: 23 µg/L

Lowest-Observed-Effect-Concentration: > 23 µg/L

Maximum Acceptable Toxicant Concentration: > 23 µg/L

21-Day EC50 (Immobility): > 23 µg/L

95% Confidence Interval: Not Calculable

21-Day EC50 (Reproduction): > 23 µg/L

95% Confidence Interval: Not Calculable

Description of key information

No effects of Brominated Epoxy on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through test conditions at the highest solubility level were observed.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
23 µg/L

Additional information