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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Feb 1997 to 10 Feb 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)
Version / remarks:
U.S. EPA Series 72 (72-3 c) of Pesticide Assessment Guidelines, FIFRA Subdivision E Hazard Evaluation: Wildlife and Aquatic Organisms; 1982
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)
Version / remarks:
U.S. Environmental Protection Agency Standard Evaluation Procedure, Acute Toxicity Test for Estuarine and Marine Organisms (Shrimp 96-Hour Acute Toxicity Test); 1985
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard E729-88a, Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Water samples were collected from one replicate test chamber of the low and high treatment groups prior to the test (pre-test) to determine if the test substance had reached equilibrium in the diluter system. Samples were also collected from each replicate test chamber of each treatment and the control group at the beginning and end of the test to measure concentrations of the test substance. Samples were collected from mid-depth of the test chambers and placed in glass scintillation vials. Samples from the replicates at test initiation and the replicates at test termination were analyzed immediately without storage. Samples not analyzed were held as backups.
Vehicle:
no
Details on test solutions:
One stock solution was prepared for each of the five concentrations tested. The first stock was prepared by dissolving the test substance in dilution water (filtered saltwater) at a concentration of 1.25 mg a.i./mL. The stock solution was stirred with an electric mixer to aid solubilization of the test substance. Aliquots of the stock solution were proportionally diluted with dilution water to prepare four additional stock solutions at concentrations of 0.750, 0.450, 0.270 and 0.162 mg a.i./mL. The five stocks were injected into the diluter mixing chambers (at a rate of 1.2 mL/minute) where they mixed with dilution water (at a rate of 100 mL/minute) to achieve the desired test concentrations. After mixing, the test solutions appeared clear and colourless.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
TEST ORGANISM
- Common name: Saltwater mysid
- Source: Mysids used in the test were obtained as juveniles from cultures maintained by Wildlife International Ltd., Easton, Maryland.

CULTURING AND HOLDING
- Culturing: Adult mysids were cultured in water from the same source and at approximately the same temperature as used during the test.
- Holding period conditions: During the holding period, the adults showed no signs of disease or stress. During the 14-day holding period preceding the test, water temperatures ranged from 24.8 to 25.3 °C. The pH of the water ranged from 7.9 to 8.0, salinity ranged from 2.0 to 2.1 % (parts per thousand) and dissolved oxygen ranged from 6.6 to 7.0 mg/L.
- Feeding: Yes, the juvenile mysids were fed live brine shrimp (Artemia sp.) nauplii daily during the test to prevent cannibalism.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24.7 - 25.0 °C
pH:
8.2 - 8.3
Dissolved oxygen:
5.9 - 6.5 mg O2/L
Salinity:
2%
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1.9, 3.2, 5.4, 9.0 and 15 mg a.i./L (based on a range-finding study; not further specified).
- Mean measured concentrations: An overview of the measured concentrations is presented in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test compartments were constructed from 500-mL glass beakers approximately 8 cm in diameter and 13 cm in height. Nylon mesh screen was attached to an opening on each side of the beakers to allow water to flow in and out of the test compartments. The compartments were suspended in 8-L Teflon®-lined stainless steel chambers filled with approximately 6.5 L of test water. The depth of the test water in a representative chamber was approximately 18 cm, whereas the depth of the test water in each test compartment was approximately 9.1 cm.
- Type: Closed, the water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross-contamination.
- Aeration: Yes, with spray nozzles
- Introduction of the individuals: At test initiation, the juvenile mysids were carefully collected from cultures and transferred to glass beakers. Mysids were transferred from the beakers to the test compartments using a wide-bore pipette. Juvenile mysids are relatively fragile and were, therefore, handled as little as possible.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- Type of flow-through (e.g. peristaltic or proportional diluter): A continuous-flow diluter was used to deliver each concentration of the test substance and a negative (dilution water) control. A peristaltic pump (Cole-Parmer Instrument Company) was used to deliver the five test substance stock solutions into mixing chambers assigned to each treatment. The stock solutions were mixed with dilution water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters. The flow of test water from each mixing chamber was split and allowed to flow into replicate test chambers. The proportion of test water that was split into each replicate was checked prior to the test to ensure that flow rates varied by no more than ±10% of the mean for the two replicates. The diluter was adjusted so that each test chamber received approximately 11.1 volume additions of test water every 24 hours. The stock solution delivery pump and the dilution water rotameters were calibrated before the test, and the general operation of the diluter was checked visually two times per day during the test and once on the last day of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity of approximately 2.0% with well water. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25μm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered to remove microorganisms and particles.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Lightning: Fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone® 50).
- Photoperiod: A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. 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: Light intensity at test initiation was approximately 150 lux at the surface of the water.

WATER QUALITY MEASUREMENTS
- Temperature: Temperature was measured in each test chamber at the beginning and end of the test using a hand-held thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ER/C Recorder. The target test temperature during the study was 25 ± 1 °C.
- Dissolved oxygen and pH: Dissolved oxygen and pH measurements were made on water samples collected from alternate replicate test chambers at test initiation and at approximately 24-hour intervals thereafter. Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 lB dissolved oxygen meter.
- Salinity: Salinity was measured in the dilution water at test initiation. Salinity was measured using an Aquafauna Bio-Marine, Inc. refractometer.

EFFECT PARAMETERS MEASURED: mortality, sub-lethal effects
Observations were made to determine the number of mortalities. The number of individuals exhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24, 48, 72, and 96 hours after test initiation.

VEHICLE CONTROL PERFORMED: no
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
6.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% C.L.: 5.8 - 8.4 mg a.i./L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
Mysids in the negative control group appeared healthy and normal throughout the test. After 96-hours of exposure, mysid mortality increased in a dose-response manner. In the 2.0, 3.3, 6.2, 9.2 and 16 mg a.i./L treatment groups the mortality was 5, 10, 40, 55 and 100%, respectively.
Reported statistics and error estimates:
When the dose response pattern allowed for the calculation of an LC50 value, the data were analyzed using the computer program of C.E. Stephan. The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, or binomial probability with nonlinear interpolation. In this study, the probit method was used to evaluate mortality at 48 and 72 hours and the moving average method was used to evaluate mortality at 96 hours. The no mortality concentration, NOEC and 24-hour LC5O were determined by visual interpretation of the mortality and clinical observation data.

Table: cumulative Percent Mortality and Treatment-Related Effects 

Mean Measured Test Concentration (mg a.i./L)

Replicate

No. Exposed

4 Hours

 

 

24 Hours

 

 

48 Hours

 

 

72 Hours

 

 

96 Hours

 

 

Cumulative Percent Mortality

No. Dead(1)

Effects(2)

No. Dead

Effects

No. Dead

Effects

No. Dead

Effects

No. Dead

Effects

Negative Control

A

10

0

10 AN

0

10 AN

0

10 AN

0

10 AN

0

10 AN

0

B

10

0

10 AN

0

10 AN

0

10 AN

0

10 AN

0

10 AN

2.0

A

10

0

10 AN

0

10 AN

0

10 AN

0

10 AN

1

9 AN

5

B

10

0

10 AN

0

10 AN

0

10 AN

0

10 AN

0

10 AN

3.3

A

10

0

10 AN

0

10 AN

1

9 AN

1

9 AN

1

8 AN; 1E

10

B

10

0

10 AN

1

9 AN

1

9 AN

1

9 AN

1

9 AN

6.2

A

10

0

10 AN

0

8 AN; 2E

1

8 AN; 1E

3

2 AN; 5E

5

1 AN; 4E

40

B

10

0

10 AN

0

9 AN; 1E

0

9 AN; 1E

2

1 AN; 7E

3

1AN; 6E

9.2

A

10

0

10 AN

1

7 AN; 2E; 1MAD

4

4AN; 2E

5

5E

6

4E

55

B

10

0

10 AN

0

7 AN; 3E

2

4 AN;4E

3

3 AN; 4E

5

2 AN; 3E

16

A

10

0

10 AN

2

8E

4

6E

8

2E

10

--

100

B

10

0

10 AN

2

1 AN; 7E

8

2E

9

1E

10

--

1) Cumulative number of dead fish

2) Observed Effects: AN = Appears Normal; E = Erratic Swimming; MAD = Missing and Assumed Dead.

Validity criteria fulfilled:
not specified
Conclusions:
The 96-hour LC50 value for saltwater mysids exposed to the test substance was 6.9 mg a.i./L. The 95% confidence limits were 5.8 and 8.4 mg a.i./L. Based on the mortality and observation data, The 96-hour no mortality concentration and the NOEC were < 2.0 mg a.i./L.
Executive summary:

The acute toxicity to saltwater invertebrates was determined in a study according to FIFRA 72-3 c and in compliance with GLP criteria. In this study saltwater mysid (M. bahia, 20 per concentration) were exposed to nominal concentrations of 0 (control), 1.9, 3.2, 5.4, 9.0 and 15 mg active ingredient (a.i.)/L for 96 hours under flow-through conditions. Analytical confirmation of nominal test concentrations showed that all test concentrations remained well within ±20% of nominal concentrations throughout the test. Mortality was recorded after approximately 4, 24, 48, 72, and 96 hours exposure. After 96-hours of exposure, mysid mortality increased in a dose-response manner. In the 2.0, 3.3, 6.2, 9.2 and 16 mg a.i./L treatment groups the mortality was 5, 10, 40, 55 and 100%, respectively. Based on these findings the 96-h LC50 value was determined at 6.9 mg a.i./L (95% C.L.: 5.8 - 8.4 mg a.i./L). The NOEC was set < 2 mg a.i./L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Jun 2000 to 9 Jun 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
test performed on Ephemeroptera Cloeon sp. nymphs. Minor deviations are summarized in 'Any other information on materials and methods incl. tables'
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
yes
Remarks:
test performed on Ephemeroptera Cloeon sp. nymphs. Minor deviations are summarized in 'Any other information on materials and methods incl. tables'
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
1996
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: US EPA Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. EPA 540/9-86-141, Guideline No.: 72-2.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analysis of test substance at 0 and 48 hours using HPLC.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution with a nominal concentration of 10 mg a.i./L was prepared by dissolving 10.1 mg of the test item completely in 1000 mL of dilution water by stirring. Using this stock solution, the nominal test concentrations as stated above were prepared by dilution of requisite volumes of stock solution with dilution water. Test solutions were added to the test vessels and the Cloeon added.
Test organisms (species):
other: Cloeon sp.
Details on test organisms:
TEST ORGANISM
- Common name: Ephemeroptera (mayfly nymph)
- Source: Field collected from pond, Stein, Switzerland
- Justification for species other than prescribed by test guideline: Cloeon sp. is a recommended species other than crustatcean for aquatic invertebrate hazard assessment for pesticides (OECD 11 Document), and demonstrates to be the most sensitive aquatic species for the test substance.
- Age: Collected at unknown age, Nymphs exposed
- Feeding during test: No

ACCLIMATION
- Acclimation period: Acclimated to test conditions for at least 20 hours
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
366 mg/L as CaCO3
Test temperature:
18.9 - 19.0°C
pH:
8.2 to 8.5
Dissolved oxygen:
87 to 96%
Nominal and measured concentrations:
- Nominal concentrations: 0 (water control), 3.1, 6.3, 13, 25, 50 and 100 µg a.s./L
- Measured concentrations at t= 0h: 92 to 104% of the nominal values
- Measured concentrations at t=48h: 91 to 100 % of the nominal values
An overview of the measured concentrations relative to the nominal values is presented in 'Any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type: Not specified
- Fill volume: 200 mL
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Pond water filtered to 90 µm and ultrasonicated for around 3 minutes.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: Fluorescent light, 16 hours light and 8 hours dark, with 30 minute dawn and dusk periods.
- Light intensity: Not reported

WATER QUALITY MEASUREMENTS
- pH and dissolved oxygen: The pH and dissolved oxygen were measured at the start and end of the test for each test concentration and the control.
- Temperature: The temperature was measured in the control at the start and end of the test.

EFFECT PARAMETERS MEASURED: immobility
Immobility of the ephemera was determined by visual observations after 24 and 48 hours of exposure. Organisms unable to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
14 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% C.L.: 11 - 17 µg a.i. /L
Details on results:
- Immobility: There was no immobility observed in the dilution water control throughout the test. Below a concentration of 6.3 µg/L no immobility was observed. throughout the test, After 24 hours 30, 60, 100 and 100% of the Cloeon sp nymphs were found immobile at nominal concentrations of 13, 25, 50 and 100 µg/L, respectively. After 48 hours immobility was observed in 50, 90, 100 and 100% of the nymphs at these concentrations, respectively. Additional details are provided in 'any other information on results incl. tables'.
Reported statistics and error estimates:
Immobilisation of the Cloeon sp. in the time period specified and was calculated by the Probit method at 24 and 48 hours.

Table: Absolute and relative immobilization data on 24 and 48 hour timepoints

Nominal concentration

Immobilised Cloeon sp. after 24 hours

Immobilised Cloeon sp. after 48 hours

(µg a.s./L)

Number

%

Number

%

0 (control)

0

0

0

0

3.1

0

0

0

0

6.3

0

0

0

0

13

6

30

10

50

25

12

60

18

90

50

20

100

20

100

100

20

100

20

100

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Conclusions:
Immobilization was recorded at exposure concentrations of 13 µg a.s./L and higher. The 48-h EC50 was calculated to be 14 µg a.s./L.
Executive summary:

The acute toxicity to nymphs of the ephereropteran Cloeon sp. was determined under static conditions. There is no agreed testing guideline for non-standard test species, however this study complies with the current validity criteria for the acute toxicity testing with Daphnia magna (OECD TG 202) and was according to GLP. Mayfly nymphs were exposed for 48 hours to a range of nominal concentrations of 0 (control), 3.1, 6.3, 13, 25, 50 and 100 µg a.s./L. At the start of the test, the measured concentrations were in the range 92 to 104% of the nominal values and at the end of the test were in the range 86 to 100%. Immobility of the ephemera was determined by visual observations after 24 and 48 hours of exposure. Organisms unable to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. Below a concentration of 6.3 µg/L no immobility was observed. throughout the test, After 24 hours 30, 60, 100 and 100% of the Cloeon sp nymphs were found immobile at nominal concentrations of 13, 25, 50 and 100 µg/L, respectively. After 48 hours immobility was observed in 50, 90, 100 and 100% of the nymphs at these concentrations, respectively. Based on nominal concentrations, the 48-h EC50 was 14 µg a.s./L.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

The freshwater 48-h EC50 value is 14 µg/L in Ephemeroptera Cloeon sp., OECD 202, Knauer 2000.

The saltwater 96-h LC50 value is 6.9 mg/L in mysid shrimp (Mysidopsis bahia), OECD 202, Drottar 1997.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.014 mg/L
EC50/LC50 for marine water invertebrates:
6.9 mg/L

Additional information

Table: Overview of available data on the short-term toxicity to aquatic invertebrates

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Ephemeroptera Cloeon sp.

OECD 202/ GLP

48-h EC50

14 µg a.i./ L

Static regime. Measured concentrations remained within 20% of nominal.

Tested in non-standard species. Some test conditions were not reported. The measured hardness of the medium was outside of the recommended range. The validity criteria were met.

Knauer, 2000

Daphnia magna

(freshwater)

OECD 202/ GLP

48-h EC50

>100 mg a.i./L

Static regime. Measured concentrations remained within 20% of nominal.

Some test conditions were not reported. The measured hardness of the medium was outside of the recommended range. The validity criteria were met.

Neumann, 1996

Mysidopsis bahia

(saltwater)

FIFRA 72-3 c

96-h LC50

6.9 mg a.i./L

Flow-through regime. Measured concentrations remained within 20% of nominal.

Drottar, 1997

Crassostrea virginica

(saltwater)

FIFRA 72-3 b / GLP

96-h EC50

> 119 mg a.i./L

Flow-through regime. Measured concentrations remained within 20% of nominal. Additional saltwater species for the refinement of the aquatic hazard assessment.

Drottar, 1997