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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: US EPA TSCA as cited Fed. Register, Sept. 27, 1985
Deviations:
yes
Remarks:
Historical TOC used instead of measured, centrifugation was used instead of filtration, Diluter calibration was not checked at study determination but analytical determinations showed consistency throughout 96 hours. Deviations have no effect on results.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Preliminary test:
- Nominal concentrations: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L
- Definitive test: 0 through 48 hours
- Nominal: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L
- Mean measured (standard deviation): 0.59 (0.055), 0.38 (0.022), 0.21 (0.0098), 0.14 (0.0075), 0.076 (0.0041) mg A.I./L

- Sampling method: with volumetric pipette from midpoint of the aquarium. Water samples removed from both replicate vessels of control and high, middle and low test concentrations one day prior to definitive exposure to analyze and assure that appropriate test conditions being maintained. During in-life phase, water samples removed and analyzed on days 0, 1 and 2 from both replicates and controls. Quality assurance samples also prepared and analyzed in parallel with test samples. Concentrations of quality assurance samples were blind to the analyst.
- Sample storage conditions before analysis: centrifugation within 30 minutes of sampling
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution containing 2.1 mg A.I./mL of 2,6 DTBP prepared by mixing 0.1001 grams of nonlabelled test material and 5.04 mg of 14C-labelled 2,6 DTBP in nanograde acetone to a total volume of 50 ml. A peristaltic pump was then used to deliver the 2.1 mg A.I./mL stock solution to the diluter system where it was diluted with dilution water (60% dilution factor) to provide the desired exposure concentration range. A similar system was used to deliver acetone to the solvent control aquaria.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 48 uL/L maximum
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no evidence of undissolved material
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: cultured at Springborn Life Sciences, Wareham, Massachusetts
- Age at study initiation (mean and range, SD): < or = 24 hours
- Feeding during test: not during 48 hour definitive exposure


ACCLIMATION
- Acclimation conditions (same as test or not): Generally the same
- Light intensity 30 to 70 footcandles
- Photoperiod 16 hours light/8 hours darkness
- Culture water: fortified well water based on ASTM 1980 for hard water and filtered through Amberlite ZAD-7 resin colu mn to remove organic contaminants with the following properties:
-total hardness and alkalinity ranges (as CaCO3) of 160-180 mg/L and 110-130 mg/L
- pH 7.9 to 8.3; temperature 20 +/- 1 degree C
- dissolved oxygen concentration > 60% saturation
- specific conductivity range of 400 to 600 micromhos per centimeter
- Type and amount of food: Green algae (Ankistrodesmus falcatus) and yeast suspension supplemented with Selco dietary supplement twice weekly
- Feeding frequency: once daily


Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
170 to 180 umhos/cm
Test temperature:
18 to 22 degrees C in continuous monitoring of one replicate of the solvent control
pH:
8.0 to to 8.4
Dissolved oxygen:
7.9 to 8.3 mg/L
Nominal and measured concentrations:

Preliminary test:
- Nominal concentrations: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L

Definitive test: 0 through 48 hours
- Nominal: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L
- Mean measured (standard deviation): 0.59 (0.055), 0.38 (0.022), 0.21 (0.0098), 0.14 (0.0075), 0.076 (0.0041) mg A.I./L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Type: open aquaria, glass test vessels, flow through system
- Material, size, headspace, fill volume: Glass test vessels maintained constant test water volume of 1.8 L and a solution depth of approximately 13 mm
- Type of flow-through: Intermittent flow proportional diluter (Mount and Brungs, 1967).
- Renewal rate of test solution (frequency/flow rate): During each cycle of the diluter system, approximately 50 mL of exposure solution was delivered to each replicate test vessel. The system cycled about 216 times per day. Each exposure vessel received about 6 solution volume replacements per day.
- No. of organisms per vessel: 10 daphnids per exposure vessel
- No. of vessels per concentration (replicates): two for each treatment level
- No. of vessels per control (replicates): two for each control
- No. of vessels per vehicle control (replicates): two for each vehicle control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: fortified well water based on ASTM 1980 for hard water and filtered through Amberlite ZAD-7 resin colu mn to remove organic contaminants with the following properties:
-total hardness and alkalinity ranges (as CaCO3) of 160-170 mg/L and 120-130 mg/L
- pH 8.1 to 8.3; temperature 20 +/- 1 degree C
- dissolved oxygen concentration > 60% saturation
- specific conductivity range of 500 micromhos per centimeter
- Total organic carbon: historically 4.3 ppm
- Particulate matter: filtered through Amberlite XAD-7 resin column ot remove any potential organic contaminants
- Metals, pesticides, chlorine: checked on periodic basis at laboratory
- Culture medium different from test medium: same source

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light/ 8 hours of dark
- Light intensity: 40 to 90 footcandles at surface provided by Cool White and Gro-Lux fluorescent lights

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization of daphnids recorded at 24 and 48 hours during exposure. Biological observations such as abnormal appearance or behavior, and observations of the physical characteristics of the test solution were made and recorded at test initiation and at 24 and 48 hours.

TEST CONCENTRATIONS
- Range finding study: Nominal concentrations: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L
- Definitive Test concentrations: Nominal: 1.0, 0.60, 0.36, 0.22, 0.13 mg Active Ingredient/L
- Results used to determine the conditions for the definitive study: At preliminary test termination, 65% of the daphnids were immobilized at 1.0 mg A.I./L. No significant immobilizations were seen with exposures of 0.60 mg A.I./L but lethargy and erratic swimming seen in treatment levels equal or greater than 0.22 mg A.I./L.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: Measurement based upon non-linear interpolation with 95% confidence intervals calculated by binomial probability
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.59 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: Measurement empirically estimated as greater than the highest mean measured concentration
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
0.076 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: See table 4. At high concentration, all daphnids immobilized at 48 hours. Less than 10% immobilization in daphnids exposed to remaining concentrations. Lethargy observed in daphnids exposed to 0.14 mg A.I/L and higher.
- Mortality of control: 5% in solvent control at 24 and 48 hours; 0% in control replicates
- Exposure solution observations: clear, colorless with no visible signs of undissolved materials

Reported statistics and error estimates:
The mean measured concentrations (days 0, 1 and 2) and corresponding effect (immobilization) data were used to estimate the 24 hour and 48 hour median effect concentrations (EC50) and 95% confidence intervals. If at least one test concentration caused immobilization of greater than or equal 50%, a computer program was used to calculate the EC50 values, and 95% confidence intervals. Three statistical methods were available: moving average angle analysis, probit analysis, and non-linear interpolation with 95% confidence intervals calculated by binomial probability. The selection of the reported EC50 value and confidence intervals was based on examination of the data, and the results of the computer analysis. Selection criteria included the establishment of a concentration-effect relationship, the number of concentrations causing partial response, and the span of responses bracketing the EC50. If two or more methods produced acceptable results, the method with the lowest confidence interval was selected.

Measured Concentrations (based on Radiometric Analyses) of 2,6-DTBP Solutions during the 48 hour Flow-through Toxicity test with Daphnids (Daphnia magna) (Table 2)

Measured concentrations (mg A.I./L)

Nominal

(mg A.I./L)

0 hour

A

0 hour

B

24 hour

A

24 hour

B

48 hour A

48 hour B

Mean (Standard Deviation)

1.0

0.51

0.55

0.57

0.58

0.66

0.64

0.59 (0.055)

0.60

0.34

0.36

0.37

0.39

0.39

0.40

0.38 (0.022)

0.36

0.21

0.20

0.21

0.22

0.22

0.22

0.21 (0.0098)

0.22

0.13

0.13

0.14

0.15

0.15

0.15

0.14 (0.0075)

0.13

0.073

0.073

0.075

0.074

0.079

0.083

0.076 (0.0041)

Solvent control

< 0.023

< 0.023

< 0.023

< 0.023

< 0.023

< 0.023

----

Control

< 0.023

< 0.023

< 0.023

< 0.023

< 0.023

< 0.023

----

Concentrations Tested and Corresponding Cumulative Number of Immobilized Organisms during the 48 hour Exposure of Daphnids to 2,6 DTBP (Table 3)

Cumulative Number of Immobilized Organisms (%)

Mean Measured Concentration (S.D.)

24 hour A

24 hour B

24 hour Mean

48 hour A

48 hour B

48 hour Mean

0.59 (0.055)

0

0

0 abc

100

100

100

0.38 (0.022)

0

10

5 ab

10

10

10 cd

0.21 (0.0098)

0

0

0ab

10

0

5 b

0.14 (0.0075)

0

0

0 e

0

0

0 b

0.076 (0.0041)

0

10

5

0

10

5

Solvent Control

0

10

5

0

10

5

Control

0

0

0

0

0

0

a Several surviving daphnids on surface of test solution

b Several surviving daphnids on bottom of vessel

c All surviving daphnids lethargic

d All surviving daphnids on bottom of vessel

e One of the surviving daphnids on surface of test solution

The EC50 values (95% confidence interval) and No Observed Effect Concentration for Daphnids (Daphnia magna) exposed to 2,6-DTBP for 48 hours (Table 4)

Confidence Interval

EC50 (mg A.I./L)

Lower (mg A.I./L)

Upper (mg A.I./L)

24-hour a

> 0.59

----

----

48 hour b

0.45

0.38

0.59

No Observed Effect Concentration Through 48 hours = 0.076 A.I./L

a EC50 value empirically estimated as greater than highest mean measured concentration

b EC50 value estimated by nonlinear interpolation; 95% confidence interval by binomial probability

Validity criteria fulfilled:
yes
Executive summary:

Daphnia magna were exposed to 2,6-di-tert-butylphenol under flow through conditions to determine the 48 hour EC50 values for immobilization of the test organisms. Twenty organisms (10 per replicate) were exposed in two replicate test vessels to five concentrations of test article, or a dilution water control, or a solvent control solution for 48 hours. Nominal concentrations of 1.0, 0.60, 0.36, 0.22, and 0.13 mg A.I./L were maintained by introducing approximately 6 aquarium volumes per day of newly prepared test solution via an intermittent-flow proportional diluter. Each replicate solution was sampled and analyzed for 2,6 DTBP as active ingredient on days 0, 1 and 2 (termination) of the exposure period. Based on the analyses, the mean measured concentrations were 0.59, 0.38, 0.21, 0.14, and 0.076 mg A.I./L. No evidence of undissolved material was noted. Biological observations were made and recorded at test initiation, and every 24 hours until the test was terminated. At the highest concentration, all daphnids were immobilized after 48 hours. Immobilization was 10% or less in daphnids exposed to the remaining concentrations. Sublethal effects (such as lethargy) was observed in daphnids exposed to 0.14 mg A.I./L. EC50 values (based on mean measured concentrations of 2,6 -DTBP as active ingredient) were greater than 0.59 mg A.I./L at 24 hours and 0.45 (0.38 -0.59) for 48 hours. The No Observed Effect Concentration through 48 hours was 0.076 mg A.I./L. The 24 hour EC50 was empirically estimated as greater than the highest mean measured concentration. The 48 hour EC50 was estimated by nonlinear interpolation and the 95% confidence interval calculated by binomial probability.

Description of key information

48h-EC50 Daphnia magna: 0.45 mg/L

96h EC50 Gammarus fasciatus: 0.6 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.45 mg/L

Additional information

Three acute toxicity invertebrate studies were performed, two using Daphnia magna and one using Gammarus fasciatus. The lowest endpoint (EC50) was found to be in Daphnia magna, 0.45 mg/L (Suprenant D.C. (1989)). The study was performed under flow-through conditions, the concentrations were based on mean measured concentrations and the quality criteria were met. Therfore, this study is considered relevant and reliable for the risk assessment. A supporting study with Gammaurs fasciatus with a 96h EC50 of 0.6 mg/L is available.