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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:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Examination/Sampling:
Survival of adult daphnids was determined on test days 0, 1, 2, 4, 7, 9, 11, 14, 16, 18 and 21. Measurements of offspring production were made on day 7 and three times per week through test termination (day 21). At each observation interval, the offspring were removed, counted and discarded. At test termination (day 21), the length and weight of each surviving adult daphnid were measured.

Monitoring of test substance (TS) concentration:
Samples were removed from each treatment level and control on days 0, 2, 7, 14 and 21 and analyzed for total PBO concentrations. All exposure solution samples were analyzed by high performance liquid chromatographic procedures.
Vehicle:
yes
Details on test solutions:
Vehicle Acetone 0.0017 % (v/v)

Vehicle control performed
A 0.41 mL/mL solvent control solution was prepared by diluting 10.2 mL acetone with distilled water to a volume of 25 mL in a volumetric flask. The syringe delivered the 41% solvent control stock solution to the solvent control aquaria which contained an amount of solvent equivalent to the maximum amount of solvent present in any test concentration.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain / Clone: Daphnia magna
- Source: From laboratory cultures maintained at Springborn Laboratories, Inc., Wareham, Massachusetts
- Age: Less than or equal to 24 hours
- Breeding method: The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light at an intensity of 24 to 38 footcandles at the surface of the culture solutions was provided by Durotest VitaLite fluorescent bulbs. Culture vessels were maintained in a temperature controlled water bath designed to maintain solution temperatures at 20  2°C.
- Kind of food Combination of a trout food suspension and a unicellular green algae (Ankistrodesmus falcatus).
- Amount of food: Not stated
- Feeding frequency: Once daily in the cultures. During exposure three times daily on weekdays and twice daily on weekends.
- Pretreatment: No
- Feeding of animals during test: Yes. Additional feeding with a dietary supplement, Selco (a commercial mixture of proteins and fatty acids, 0.6 mg/mL)
Test type:
flow-through
Total exposure duration:
21 d
Hardness:
160 – 180 mg/L as CaCO3
Test temperature:
19 - 21°C
pH:
7.9 – 8.3
Dissolved oxygen:
8.1 – 8.3 mg/L
Details on test conditions:
TEST SYSTEM
Test type: flow-through
Renewal of test solution:approx. 6 volume replacements every 24 hours
Volume of test vessels:1.4 L
Volume/animal: 0.14 L
Number of animals/vessel:10
Number of vessels/ concentration: 4
Test performed in closed vessels due to significant volatility of TS:No

TEST MEDIUM / WATER PARAMETERS (dilution water)
Source: Hard fortified well water
Alkalinity: 110 – 130 mg/L as CaCO3
Hardnes: 160 – 180 mg/L as CaCO3
pH: 7.9 – 8.3
Ca / Mg ratio: Not stated
Na / K ratio: Not stated
Oxygen content: 8.1 – 8.3 mg/L
Conductance: 400 – 600 µmhos/cm
TOC: 0.43 and 0.79 mg/L
Holding water different from dilution water: Yes

OTHER TEST CONDITIONS
Adjustment of pH: No
Aeration of dilution water: No
Quality/Intensity of irradiation: Durotest Vitalite® fluorescent lights centrally located above the test aquaria at a range of 38 to 56 footcandles (410 to 600 lux)
Photoperio: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED:
Immobility (e.g. mortality) of adult daphnids
Number of living young daphnids
Number of dead young daphnids
Appearance and behaviour of adult daphnids (At test termination (day 21))
growth (total body length and dry weight) (At test termination (day 21))

VEHICLE CONTROL PERFORMED: yes. A 0.41 mL/mL solvent control solution was prepared by diluting 10.2 mL acetone with distilled water to a volume of 25 mL in a volumetric flask. The syringe delivered the 41% solvent control stock solution to the solvent control aquaria which contained an amount of solvent equivalent to the maximum amount of solvent present in any test concentration.

RANGE-FINDING STUDY
Range finding test: Performed
Concentrations: 31, 63, 130, 250 and 500 µg PB200/L under static conditions
Number/ percentage of animals showing adverse effects: Following 11 days of exposure, survival of 100, 100, 95, 95, and 75 % was observed in the 31, 63, 130, 250 and 500 µg PB200/L treatment levels respectively. Reproduction after 11 days of exposure was 40, 45, 24, 38 and 2 offspring per female in the 31, 63, 130, 250 and 500 µg PB200/L treatment levels respectively. In the control the mean percent survival was 95 %. Control reproduction averaged 44 offspring per female.
Nature of adverse effects: Not stated
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
30 µg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
47 µg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 650 µg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
38 µg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Details on results:
RESULTS TEST SUBSTANCE
Initial concentrations of test substance: 47, 94, 190, 380 and 750 µg PBO/L under flow-through conditions
Actual concentrations of test substance: 30, 47, 95, 210 and 650 µg PBO/L
Effect data: After a 21-day exposure to PB200, the 21-day EC50 was estimated to be >650 µg PBO/L, above the highest tested mean measured concentration. Reproduction among organisms exposed to the 47, 95 and 210 µg PBO/L treatment levels was 180, 159 and 33 offspring per female, respectively, and was significantly reduced as compared to the performance of the pooled control organisms (201 offspring per female). The LOEC was determined to be 47 µg PBO/L and the NOEC was determined to be 30 µg PBO/L. Utilizing these data, the MATC for PBO and Daphnia magna was established to be 38 µg PBO/L (>30µg/L and < 47 µg/L).
Concentration/response curve: see the graphic "CONCENTRATION-RESPONSE CURVE" attached in background material
Other effects: After 21 days of exposure mean total length of organisms exposed to the 210 µg PBO/L treatment level was 5.1 mm and statistically reduced as compared to the mean total length of the pooled control organisms (5.2). Total length of the organisms of the other treatment levels and also the mean dry weight of the organisms in all treatment levels showed no significant difference as compared to the pooled control organisms.

For the results of controls see the tables "OFFSPRING PRODUCED PER FEMALE DAPHNID" and "PERCENT SURVIVAL OF PARENTAL DAPHNIDS" attached in background material.
Reported statistics and error estimates:
All statistical analyses were conducted at the 95% level of certainty except in the case of the Shapiro-Wilks Test and the Bartlett’s Test, in which the 99% level of certainty was applied. Data representing endpoints estimated as a proportion were transformed (e.g., arcsine square root percentage) prior to analysis. Student ‘s t-Test was used to evaluate the endpoints of survival, reproduction and growth and to compare the performance of the dilution water control organisms with that of the solvent control organisms. If analyses demonstrated no significant difference between the two control groups, all subsequent analyses were performed using pooled control data. The Shapiro-Wilks Test for normality was used to compare the observed sample distribution with a normal distribution for all endpoints. If the data are not normally distributed, then a non-parametric procedure is used for subsequent analyses. As a check on the assumption of homogeneity of variance, data for each endpoint were analyzed using Bartlett’s Test. Survival, reproduction and mean body weight met the assumptions for homogeneity of variance, therefore, statistical comparisons were performed using the Williams’ Test. Mean total body length did not meet the assumptions for homogeneity of variance therefore, the Wilcoxon Rank Sum Test was used. Treatment levels at which significant adverse effects were observed were excluded from statistical analysis of daphnid reproduction and growth.

Computer program by Gulley, D.D., Boetler, A.M. and Bergman, H.L. 1991 Toxtat Release 3.3 University of Wyoming, Laramie, Wyoming, was used to perform the computations.
Validity criteria fulfilled:
yes
Remarks:
according to OECD Guideline 211
Conclusions:
Validity criteria can be considered as fulfilled. (see validity criteria summarized in the Table "VALIDITY CRITERIA ACCORDING TO OECD GUIDELINE 211" attached in background material
Reliability 1
Deficiencies No

Description of key information

Key value for chemical safety assessment

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

Additional information