Diquat dibromide

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2013 to 9 Jul 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA OCSPP Draft Guideline 850.1770

Version / remarks:

2009

Deviations:

no

Principles of method if other than guideline:

The methods were designed to meet the testing requirements in the EPA document entitled "Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, 2nd Edition", test method 100.4 (U.S. EPA, 2000).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Dosed sediment: Dosed sediments were sampled during the mixing/equilibration period prior to the allocation of the sediments into the replicate exposure vessels. In addition, subsamples of the dosing stock solutions used to dose the sediments were also analyzed for test substance concentration. Results of these pretest analyses were used to confirm that sufficient quantities of the test substance had been applied during the dosing process.
- Overlying water: During the in-life phase of the definitive study, all overlying water, pore water and sediment samples were removed and analyzed for the test substance concentration on test days 0 (exposure initiation), day 14 and 28 (termination of sediment phase of the exposure). On day 0, samples were removed and analyzed from replicate vessels M of all treatment levels and the control. On day 14, samples were removed and analyzed from replicate N of all treatment levels and the control, while on day 28, samples were removed and analyzed from replicate O of all treatment levels and the control.
- Pore water: Pore water samples were collected by removing the entire sediment sample from each test vessel and centrifuging for 15 to 30 minutes at approximately 10,000 g, with the exception of day 0 where samples were centrifuged at 1,200 g due to equipment failure. The resulting pore water was removed from the centrifuge tube and the appropriate sample volume measured. Sediment samples were collected from the centrifuge tube with a stainless steel spatula, following centrifugation and removal of the pore water sample. To achieve homogeneous sub-samples, sediment samples were mixed well after the removal of pore water.

Vehicle:

no

Details on sediment and application:

- Stock Solution Preparation: A 20 mg/mL primary stock solution was prepared by placing 2.4888 g of test material (1.0005 g a.i.) in a 50 mL volumetric flask and bringing it to volume with deionized water. The resulting stock solution was observed to be clear and brown in color with no visible undissolved test substance.
- Sediment Dosing Stock Solutions: Five individual dosing stock solutions were prepared in deionized water for application of the test substance to the sediment. The dosing stock preparation scheme is provided in Table 1 in 'Any other informaton on materials and methods inlc. tables'. All dosing stock solutions were observed to be clear and brown/gold in color with no visible undissolved test substance following preparation.
- Application of Test Substance to Sediment: A jar-rolling technique was used to apply the test substance to the sediment. A 20 mL volume of each dosing stock solution was applied to 3.5 kg of wet sediment (1.4959 kg dry weight of test sediment based on a percent solids value of 42.74%) in individual glass jars. The scheme of applying the test usbtance to the sediment is provided in Table 2 in 'Any other information on materials and methods incl. tables'.
The jars were sealed and positioned horizontally on the rolling mill. Each jar was then rolled for four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the jars were stored upright at 2 to 8 ºC. The sediments were allowed to equilibrate for a 20-day period in the refrigerator. Once a week during the 20-day equilibration period and prior to distribution of the sediments into the replicate test vessels, the jars were mixed on the rolling mill as previously mentioned for an additional two hours at room temperature to ensure the sediment was homogeneous. The negative control sediment consisted of 3.5 kg of unadulterated wet sediment without the addition of test substance and was equilibrated and stored with the treated sediments.

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Common name: Amphipod
- Source: Obtained from laboratory cultures maintained at the test facility
- Age of animals at beginning of exposure: Juvenile amphipods (8 days old)
- Details on collection: The test organisms were collected from reproducing adult amphipods removed from the main culture tanks 9 days prior to test initiation. The adult amphipods were placed in 9.5-L aquaria (isolation tanks) containing approximately 8 L of water. Neonate amphipods (< 24 hours old) produced by these isolated adults were then removed from the isolation tanks and pipetted into 1-L beakers containing approximately 0.80 L of laboratory dilution water. The neonate amphipods were reared under static conditions for 8 days with gentle, oil-free aeration.
- Feeding during test: During the 42-day exposure, each replicate test vessel received 1.0 mL of YCT (yeast, cereal leaves and flaked fish food suspension) daily.
- Food quality during test: Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed. Based on these analyses, longevity of the culture population, and survival of the juvenile test organisms, the food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern (ASTM, 2002).

ACCLIMATION
- Acclimation period:
- Acclimation conditions: Prior to exposure initiation, amphipods were maintained in 20-L glass aquaria containing approximately 15 L of culture water under flow-through conditions. The culture water was from the ame source as the overlying water used during the test.
- Water parameters: Dissolved oxygen ranged from 5.9 to 8.0 mg/L (72 to 100% air saturation value) and temperature ranged from 25 to 27 °C.
- Type and amount of food: Fed a combination of yeast, cereal leaves and flaked fish food suspension. Ankistrodesmus falcatus, a unicellular green algae as well as 3 drops of a 100 mg/mL flaked fish food suspension, were also provided as a supplemental food source at the start of the holding period.
- Feeding frequency: Once daily
- Health of the organisms: The test organisms appeared healthy and no mortality was observed in the juvenile test population 48 hours prior to exposure initiation.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

42 d

Exposure phase:

total exposure duration

Hardness:

36 - 48 mg/L as CaCO3

Test temperature:

- Overlying water: 22 - 24 ºC
- Pore water: 20 - 21 ºC

pH:

- Overlying water: 6.7 - 7.6
- Pore water: 5.9 - 6.3

Dissolved oxygen:

2.6 - 8.1 mg O2/L

Ammonia:

- Overlying water: 0.12 - 0.92 mg/L as N
- Pore water: 14.1 - 4.4 mg/L as N

Conductivity:

230 - 300 μS/cm

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 6.3, 13, 25, 50 and 100 mg a.i./kg dry sediment
- Measured concentration (in sediment): < LOQ (negative control), 5.6, 12, 21, 43 and 87 mg a.i./kg dry sediment, respectively. (See Table 3 in 'Any other information on materials and methods incl. tables')
- Measured concentration (in overlying water) : < LOQ (negative control), 0.0061, 0.0065, 0.017, 0.050 and 0.020 mg a.i./kg dry sediment, respectively. (See Table 4 in 'Any other information on materials and methods incl. tables')
- Measured concentration (in pore water) : < LOQ (negative control),

Details on test conditions:

TEST SYSTEM
- Test container: 300 mL glass vessels
- Sediment volume: 100 mL (approximately 4.0-cm layer) of sediment (equivalent to 125 g wet weight per vessel or 53.4 g dry weight per vessel)
- Overlying water volume: 175 mL
- Total volume: The total overlying water plus sediment volume was maintained at approximately 275 mL.

EXPOSURE REGIME
- No. of organisms per container: 10
- No. of replicates per treatment group: 12
- No. of replicates per control: 18 (12 for biological analysis, 3 for chemical analysis and 3 for pore water quality measurement)

RENEWAL OF OVERLYING WATER
- Details on volume additions: Adding two volume additions of water (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system
- Flow-rate: The water delivery system cycled approximately 7 times per day, providing approximately 350 mL per vessel every 24 hours.

OVERLYING WATER CHARACTERISTCS
- Type of water: Laboratory well water
- Total hardness: 36 - 48 mg/L (as CaCO3)
- Total alkalinity: 18 - 26 mg/L (as CaCO3)
- pH: 7.0 - 7.9
- Conductivity: 260 - 350 μS/cm
- Total organic carbon: Representative samples of the overlying water source were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the overlying water source was 1.0 and 1.7 mg/L for May and June 2013, respectively.
- Others: Representative samples of the overlying water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected in any of the water samples analyzed in agreement with ASTM guidelines (2002).
The overlying water information confirmed the acceptability of this overlying water for use in toxicity testing.

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Glen Charlie Pond, Wareham, Massachusetts.

CHARACTERIZATION OF SEDIMENT
Prior to use and characterization, the sediment was wet pressed through a 2.0-mm sieve to remove large particles and indigenous organisms.
- Organic carbon: 3.1% (based on three measurements)
- % dry weight of sphagnum moss peat:
- pH: 5.5
- % sand: 89%
- % silt: 8%
- % clay: 3%
- Percent solids: 42.74%
- Pore water ammonia concentration: 4.3 mg/L as nitrogen
- OtherL Representative samples of the sediment were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analyzed, in agreement with ASTM (2002) standard practice.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 460 - 870 lux

WATER QUALITY MEASUREMENT
- Dissolved oxygen concentration, temperature and pH: Dissolved oxygen concentration, temperature and pH were measured in the overlying water of each replicate vessel of each treatment level and the control used for biological monitoring (Replicates A through L, except at test day 29 and test termination, minus vessels C, G, H and K for the control and treatment levels ≤ 25 mg/kg nominal; minus vessel replicates A, C, G, H and K for the 50 mg/kg nominal treatment level; and minus vessel replicates B, C, G, H and K for the 100 mg/kg nominal treatment level, which were sacrificed or contained no amphipods at test day 28) during the 42-day exposure. On the remaining test days, dissolved oxygen and temperature were measured in one alternating replicate of each treatment level and control each day with the exception of measurements in the 50 mg/kg nominal treatment level on test days 31 and 32. In addition, the temperature was continuously monitored in an auxiliary vessel in the temperature controlled water bath used to house the test vesselsthroughout the study.
- Total hardness, alkalinity, conductivity and total ammonia concentration: Total hardness, alkalinity, conductivity and total ammonia concentration of the overlying water were monitored at exposure initiation, test day 28, test day 29 and at test termination (test day 42) in each treatment level and the control group from a composite sample (replicates A through L, except at test day 29 and termination, minus vessel replicates C, G, H and K for the control and treatment levels ≤ 25 mg/kg nominal; minus vessel replicates A, C, G, H and K for the 50 mg/kg nominal treatment level; and minus vessel replicates B, C, G, H and K for the 100 mg/kg nominal treatment level, which were sacrificed or contained no amphipods at test day 28).

PORE WATER
At toxicity exposure initiation, test day 14 and test day 28, ammonia (as nitrogen) concentration, temperature and pH were measured in a pore water sample of the control. In addition, redox potential (Eh) was measured at the midpoint of the sediment layer (approximately 2 cm deep) prior to removing the sediment for centrifugation at each sampling interval. These measurements were considered representative of all treatment levels and established that the sediment remained suitable to support the organism for the duration of the exposure. Pore water samples for ammonia, temperature, pH and redox potential measurements were taken from replicates P, Q and R, respectively, on days 0, 14 and 28.

EFFECT PARAMETERS MEASURED
Daily observations of organism behavior (e.g., adverse effects) were made and the physical characteristics of the test solutions were recorded.
Prior to test day 28, four of the twelve replicate vessels maintained for biological observations (replicate vessels C, G, H and K for the control and each treatment level) were randomly selected. Amphipod survival and growth (length) were determined in these test vessels on test day 28 by sieving the sediment to remove all surviving amphipods. The adults from these replicates were preserved for up to two weeks in a sugar formalin solution prior to taking images for length determination. Growth was determined by measuring body length from the base of the first antenna to the tip of the third uropod along the curve of the dorsal surface to the nearest 0.01 mm using an image analyzer.
The amphipods in the remaining replicates were also removed by sieving and survival of these organisms was recorded. The surviving amphipods from these replicates were then placed in 300 mL water-only exposure vessels containing a 3 cm x 3 cm piece of nylon screen.
Reproduction and survival of the amphipods was measured on test days 35 and 42 by removing and counting the adults and offspring in each replicate beaker. In addition, any offspring observed at the end of the sediment exposure phase (test day 28) were counted and recorded.
On day 35, adults were enumerated to assess day 35 survival and returned to their respective test vessels after reproduction had been assessed. At test termination (day 42), the adult amphipods were enumerated to assess day 42 survival and preserved in sugar formalin solution for up to two weeks prior to taking images for length determination. The number of adult males and females were determined following preservation, as well as the number of gravid females (identified by the presence of eggs within the brood pouch. Mature males were identified by the enlarged second gnathopod. Those amphipods not identified as males were recorded as female amphipods. Reproduction for both day 35 and 42 is expressed as the number of young per adult female amphipod in each replicate based on the number of females present at test day 42. The day 42 growth (measured as length) of surviving, preserved amphipods was determined after test termination and measured using the same process to measure length on individuals from test day 28.

SEDIMENT-WATER EQUILIBRATION TRIAL
- Test procedures: Prior to dosing the sediment for the definitive exposure, a sediment-pore water equilibration trial was conducted to determine the appropriate equilibration time for sediment toxicity testing with this material. During this trial, a single, nominal sediment concentration (100 mg/kg) was prepared by adding 10 mL of a 13.2 mg/mL the test substance stock solution to 2.0 kg of wet sediment in a mixing jar. The sediment/test substance was rolled for 4 hours on a rolling mill at 15 rpm and stored upright overnight at 2 to 8 ºC. One day after application, the sediment/test substance was rolled again for an additional 2 hours. After rolling, the sediment was sampled in triplicate and centrifuged at 10,000 g for 15 to 30 minutes to generate pore water samples. Pore water was decanted and analyzed by liquid chromatography with mass spectrometry (LC/MS/MS) for the test substance concentration. The rolling and sampling procedure was repeated on days 7, 14, 21 and 28.
- Results: The results of the equilibration trial demonstrated that by day 21, the sediment and pore water had reached equilibrium using empirical observations. Equilibration was defined as the initial time point at which the highest mean pore water concentration was observed and subsequently sustained for later time points. Consequently, an approximate 21-day equilibration time was used in the definitive sediment toxicity testing with this material.

Reference substance (positive control):

no

Key result

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

21 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 87 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

87 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: growth and male:female ratio

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

21 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Table 6 - Table 9 in ‘Any other information on results incl. tables’.

- Amphipod Survival and Growth (Test Day 28):
Following 28 days of exposure, amphipod survival in the control averaged 83%. During the same period, amphipod growth in the control averaged 5.33 mm per amphipod. Amphipod survival and growth observed in the control during this period met the minimum acceptance criteria established by the EPA test method 100.4 (i.e., ≥ 80% survival and mean length ≥ 3.2 mm per amphipod at test day 28). As demonstrated by the control organism performance, the exposure system provided test conditions that were appropriate for acceptable survival and growth of Hyalella azteca.
On test day 28, survival observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg a.i./kg dry sediment treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) averaged 94, 86, 97, 80 and 90%, respectively. Statistical analysis (Steel's Many-One Rank Test) demonstrated no significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control (83%). On test day 28, length among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) averaged 5.22, 5.48, 5.12, 5.32 and 5.14 mm per amphipod, respectively. Statistical analysis (Dunnett's Multiple Comparison Test) demonstrated no significant difference in length in any of the treatment levels tested compared to the control organisms (5.33 mm per amphipod).

- Amphipod Survival and Reproduction (Day 35): Following 35 days of exposure, amphipod survival in the control averaged 83%. During the same period, amphipod reproduction in the control averaged 10.8 offspring per female. Mean percent survival of 88, 78, 94, 73 and 83% was observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg a.i./kg dry sediment treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg), respectively. Statistical analysis (Steel's Many-One Rank Test) determined no significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control (83%).
The mean number of offspring per female among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) was 6.3, 9.1, 7.8, 4.9 and 5.8, respectively. Statistical analysis (Bonferroni’s Adjusted t-Test) determined a significant difference in reproduction among amphipods exposed to the 5.6, 43 and 87 mg/kg treatment levels compared to the control (10.8 offspring per female). Due to the lack of statistical significance at the 12 and 21 mg/kg treatment levels, the effect observed at the 5.6 mg/kg treatment level was determined to not be toxicant related, and was likely attributable to a delay in brood release relative to the day of enumeration. The conclusion that a reduction in the number of offspring at the 5.6 mg/kg treatment relative to the control was not toxicant related is further supported by the fact that there was not a significant difference noted after day 42. A 55% reduction in reproductive output at 43 mg/kg was observed compared to the control data; however, due to the variability observed and the lack of a monotonic dose response, these data were not considered to be appropriate for an EC50 determination. The 35-day EC50 value for reproduction was empirically estimated to be > 87 mg a.i./kg (47 mg cation/kg), the highest mean measured sediment concentration tested.

- Amphipod Survival, Growth, Reproduction and Sex Ratio (Test Day 42): Mean percent survival among amphipods in the control was 75%. Mean percent survival of 88, 76, 91, 70 and 83% was observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg), respectively. Statistical analysis (Steel's Many-One Rank Test) determined no significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control (75%). Mean growth among amphipods in the control averaged 5.78 mm per amphipod. Mean growth among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) averaged 5.64, 6.03, 5.76, 5.93 and 5.88 mm per amphipod, respectively. Statistical analysis (Bonferroni’s Adjusted t-Test) determined no significant difference in length among amphipods exposed to any of the treatment levels tested compared to the control organisms (5.78 mm per amphipod).
Reproduction among amphipods in the control averaged 16.9 offspring per female. The test method requires cumulative reproduction of ≥ 2 offspring per control female between test days 28 and 42. As demonstrated by the control organism performance, the exposure system provided test conditions that were appropriate for acceptable reproduction of Hyalella azteca. The mean number of offspring per female among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) was 13.5, 19.2, 14.2, 11.7 and 11.1, respectively. Statistical analysis (Wilcoxon's Test with Bonferroni’s Adjustment) determined a significant difference in reproduction among amphipods exposed to the 43 and 87 mg/kg treatment levels compared to the control (16.9 offspring per female). The lack of a monotonic response related to offspring production in the control and treatments ≤ 21mg/kg suggests that the reductions observed at 43 and 87 mg/kg may be attributable to the natural variability associated with offspring production in the test system, rather than a toxicant-related response.
The mean male:female ratio among amphipods in the control was 0.65. Mean male:female ratio among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels (3.0, 6.4, 11, 23 and 47 mg cation/kg) was 0.73, 1.7, 1.9, 1.6 and 1.1, respectively. Statistical analysis (Wilcoxon's Test with Bonferroni’s Adjustment) determined no significant difference in male:female ratio among amphipods exposed to any of the treatment levels tested compared to the control (0.65). Given the nature of this endpoint and the data set, an EC50 assessment was not applicable.

Reported statistics and error estimates:

Table statistical analysis in 'Any other information on materials and methods incl. tables'

Table 6. Mean percent survival of adult amphipods and mean amphipod growth (length) during the chronic exposure of amphipods (Hyalella azteca) to the test substance on test day 28

Mean Measured Sediment Concentration		Test Day 28
(mg a.i./kg)	(mg cation/kg)	Mean Percent Survival (SD^a)	Mean Length per Amphipod in mm (SD)
Control	Control	83 (34)	5.33 (0.17)
5.6	3.0	94 (9)	5.22 (0.13)
12	6.4	86 (16)	5.48 (0.19)
21	11	97 (7)	5.12 (0.31)
43	23	80 (28)	5.32 (0.13)
87	47	90 (29)	5.14 (0.28)

SD = Standard Deviation.

 

Table 7. Mean percent survival of adult amphipods and mean number of offspring released per female amphipod during the chronic exposure of amphipods (Hyalella azteca) to the test substance on test day 35

Mean Measured Sediment Concentration		Test Day 28
(mg a.i./kg)	(mg cation/kg)	Mean Percent Survival (SD^a)	Mean Number of Offspring Released per Female (SD)
Control	Control	83 (30)	10.8 (2.8)
5.6	3.0	88 (12)	6.3^b(3.5)
12	6.4	78 (18)	9.1 (2.9)
21	11	94 (7)	7.8 (3.4)
43	23	73 (30)	4.9^c(1.9)
87	47	83 (34)	5.8^c(1.1)

a SD = Standard Deviation.

b Significantly reduced compared to the control, based on Bonferroni’s Adjusted t-Test.Due to the lack of statistical significance at the 12 and 21 mg/kg treatment levels, the effect observed at this treatment level was determined to not be toxicant related.

c Significantly reduced compared to the control, based on Bonferroni’s Adjusted t-Test.

Table 8. Mean percent survival of adult amphipods, mean amphipod growth (length), mean number of offspring released per female and mean male:female ratio during the chronic exposure of amphipods (Hyalella azteca) to the test substance on test day 42

Mean Measured Sediment Concentration		Test Day 42
(mg a.i./kg)	(mg cation/kg)	Mean Percent Survival (SD^a)	Mean Length per Amphipod in mm (SD)	Mean Number of Offspring Released per Female (SD)	Mean Male:Female Ratio (SD)
Control	Control	75 (30)	5.78 (0.29)	16.9 (2.8)	0.65 (0.5)
5.6	3.0	88 (12)	5.64 (0.19)	13.5 (5.7)	0.73 (0.4)
12	6.4	76 (18)	6.03 (0.31)	19.2 (8.0)	1.7 (1.8)
21	11	91 (8)	5.76 (0.19)	14.2 (4.6)	1.9 (2.2)
43	23	70 (29)	5.93 (0.31)	11.7^b (1.9)	1.6 (1.0)
87	47	83 (34)	5.88 (0.23)	11.1^b (2.1)	1.1 (0.7)

a. SD = Standard Deviation.

b. Significantly reduced compared to the control, based on Wilcoxon's Test with Bonferroni’s Adjustment.

 

Table 9. Established endpoints for the chronic exposure of amphipods (Hyalella azteca) to the test substance applied to sediment

Day 28 – Survival and growth
Endpoint	Amphipod Survival	Amphipod Growth
LOEC	> 87 mg a.i./kg > 47 mg cation/kg	> 87 mg a.i./kg > 47 mg cation/kg
NOEC	87 mg a.i./kg 47 mg cation/kg	87 mg a.i./kg 47 mg cation/kg
LC/EC50	> 87 mg a.i./kg > 47 mg cation/kg	> 87 mg a.i./kg > 47 mg cation/kg
Day 32 – Survival and reproduction
Endpoint	Amphipod Survival	Amphipod Growth
LOEC	> 87 mg a.i./kg > 47 mg cation/kg	43 mg a.i./kg 23 mg cation/kg
NOEC	87 mg a.i./kg 47 mg cation/kg	21 mg a.i./kg 11 mg cation/kg
LC/EC50	> 87 mg a.i./kg > 47 mg cation/kg	> 87 mg a.i./kg > 47 mg cation/kg
Day 42 – Survival and growth
Endpoint	Amphipod Survival	Amphipod Growth
LOEC	> 87 mg a.i./kg > 47 mg cation/kg	43 mg a.i./kg 23 mg cation/kg
NOEC	87 mg a.i./kg 47 mg cation/kg	87 mg a.i./kg 47 mg cation/kg
LC/EC50	> 87 mg a.i./kg > 47 mg cation/kg	> 87 mg a.i./kg > 47 mg cation/kg
Day 42- Reproduction and male : female ratio
LOEC	43 mg a.i./kg 23 mg cation/kg	> 87 mg a.i./kg > 47 mg cation/kg
NOEC	21 mg a.i./kg 11 mg cation/kg	87 mg a.i./kg 47 mg cation/kg
LC/EC50	> 87 mg a.i./kg > 47 mg cation/kg	NA

NA = Not Applicable. LC/EC50 value was empirically estimated. Therefore, corresponding 95% confidence intervals could not be calculated.

 

 

 

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 42-day NOEC for reproduction was determined to be 21 mg a.i./kg sediment dw and 28-day LC50 was determined to be > 87 mg a.i./kg sediment dw.

Executive summary:

This study was performed to determine the effects of the test substance applied to sediment, on the freshwater amphipod, Hyalella azteca, under static-renewal conditions for 42 days following EPA OCSPP guideline 850.1770. It was in compliance with GLP criteria. The nominal test concentration were 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight (mean measured concentrations were 5.6, 12, 21, 43 and 87 mg a.i./kg sediment dry weight, respectively). In addition, a negative control was included in the study as well. Twelve replicates were used to evaluate biological response of the test organisms. The final three replicates were maintained for chemical analysis. Three additional control replicates were maintained for pore water quality measurements. Test vessels were maintained in a water bath at 22 to 25 °C with a 16 hour light: 8 hour dark photoperiod at a light intensity range of 460 to 870 lux. Observations of mortality and abnormal behavior were made and the physical characteristics of the sediment/overlying water were recorded.

Following 28 days of exposure, amphipod survival in the control averaged 83%. The survival observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg a.i./kg dry sediment treatment levels averaged 94, 86, 97, 80 and 90%, respectively. No significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control. On test day 28, length among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels averaged 5.22, 5.48, 5.12, 5.32 and 5.14 mm per amphipod, respectively. No significant difference in length in any of the treatment levels tested compared to the control organisms (5.33 mm per amphipod).

Following 35 days of exposure, amphipod survival in the control averaged 83%. During the same period, amphipod reproduction in the control averaged 10.8 offspring per female. Mean percent survival of 88, 78, 94, 73 and 83% was observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg a.i./kg dry sediment treatment levels, respectively. No significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control (83%). The mean number of offspring per female among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels was 6.3, 9.1, 7.8, 4.9 and 5.8, respectively. A significant difference in reproduction among amphipods exposed to the 5.6, 43 and 87 mg/kg treatment levels compared to the control (10.8 offspring per female). A 55% reduction in reproductive output at 43 mg/kg was observed compared to the control data; however, due to the variability observed and the lack of a monotonic dose response, these data were not considered to be appropriate for an EC50 determination.

Mean percent survival among amphipods in the control was 75%. Mean percent survival of 88, 76, 91, 70 and 83% was observed among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels, respectively. No significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control. Mean growth among amphipods in the control averaged 5.78 mm per amphipod. Mean growth among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels averaged 5.64, 6.03, 5.76, 5.93 and 5.88 mm per amphipod, respectively. No significant difference in length among amphipods exposed to any of the treatment levels tested compared to the control organisms (5.78 mm per amphipod).

Reproduction among amphipods in the control averaged 16.9 offspring per female. The test method requires cumulative reproduction of ≥ 2 offspring per control female between test days 28 and 42. The mean number of offspring per female among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels was 13.5, 19.2, 14.2, 11.7 and 11.1, respectively. A significant difference in reproduction among amphipods exposed to the 43 and 87 mg/kg treatment levels compared to the control. The lack of a monotonic response related to offspring production in the control and treatments ≤ 21mg/kg suggests that the reductions observed at 43 and 87 mg/kg may be attributable to the natural variability associated with offspring production in the test system, rather than a toxicant-related response.

The mean male:female ratio among amphipods in the control was 0.65. Mean male:female ratio among amphipods exposed to the 5.6, 12, 21, 43 and 87 mg/kg treatment levels was 0.73, 1.7, 1.9, 1.6 and 1.1, respectively. No significant difference in male:female ratio among amphipods exposed to any of the treatment levels tested compared to the control. Given the nature of this endpoint and the data set, an EC50 assessment was not applicable.

Based on the findings, the 42-day NOEC for reproduction, the most sensitive endpoint, was determined to be 21 mg a.i./kg sediment dw and 28-day LC50 was determined to be > 87 mg a.i./ kg sediment dw.

Description of key information

42-d NOEC = 21 mg registered substance/kg sediment dw based on reproduction, Hyalella azteca, EPA 850.1770, Bradley 2013

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

21 mg/kg sediment dw

Additional information

The effects of the test substance on the freshwater amphipod, Hyalella azteca, under static-renewal conditions for 42 days when applied to sediment was studied under GLP to EPA OCSPP guideline 850.1770. Mean measured concentrations of 5.6, 12, 21, 43 and 87 mg pure test substance/kg sediment dry weight were tested together with a negative control with two replicates each. Three replicates were maintained for chemical analysis. Three additional control replicates were maintained for pore water quality measurements.

Following 28 days of exposure, amphipod survival in the control averaged 83%. No significant difference in survival was observed among amphipods exposed to any of the test concentrations compared to the control. No significant difference in length of amphipods exposed to any test concentration occurred compared to the control organisms. No significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control after 35 days of exposure. However, a significant difference in reproduction among amphipods exposed to the 5.6, 43 and 87 mg/kg treatment levels compared to the control (10.8 offspring per female) was observed at this timepoint. A 55% reduction in reproductive output at 43 mg/kg was observed compared to the control data; however, due to the variability observed and the lack of a monotonic dose response, these data were not considered to be appropriate for an EC50 determination. After 42 days of exposure, mean percent survival among amphipods in the control was 75%. No significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the control. Furthermore, no significant difference in length among amphipods exposed to any of the treatment levels tested compared to the control organisms (5.78 mm per amphipod) was seen.
Reproduction among amphipods in the control averaged 16.9 offspring per female after 42 days, meeting the requirements of the test guideline for cumulative reproduction of ≥2 offspring per control female between test days 28 and 42. A significant difference in reproduction among amphipods exposed to the 43 and 87 mg/kg compared to the control was found. The lack of a monotonic response related to offspring production in the control and treatments ≤21 mg/kg suggested that the reductions observed at 43 and 87 mg/kg may be attributable to the natural variability associated with offspring production in the test system, rather than a substance-related response. No significant difference in male:female ratio occurred among amphipods exposed to any of the treatment levels tested compared to the control. Given the nature of this endpoint and the data set, an EC50 assessment was not applicable. The 42-day NOEC for reproduction of freshwater amphipods exposed to the substance via sediment, the most sensitive endpoint, was determined to be 21 mg pure test substance/kg sediment dw and the 28-day LC50 was determined to be >87 mg pure test substance/kg sediment dw.