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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
Study period:
03 January 2001 to 04 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Water samples were collected from each replicate test chamber at test initiation. Water samples were also collected from each test chamber on Day 1 (old and new solutions) and at test termination.
- The samples were collected from mid-depth in each test chamber, placed directly in autosampler vials, capped and analysed as soon as possible without storage.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- A primary stock solution was prepared at a concentration of 100 mg/L by volumetrically adding 18 µL of test material to 410 mL of dilution water using a gas tight syringe.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Age at study initiation: Less than 24 hours old when Neonate daphnids were obtained for testing from individual adult daphnids.
- Feeding during test: No, daphnids in the culture were fed a mixture of yeast, Cerophyll and trout chow as well as a suspension of Freshwater green algae. Adults were fed prior to test initiation but the neonates were not fed during the test.

ACCLIMATION
- Acclimation conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The 0 hours measurement for hardness was 126 mg/L as CaCO3
Test temperature:
20 ± 1 °C
pH:
8.3 to 8.5
Dissolved oxygen:
≥ 8.8 mg/L (97% of saturation)
Nominal and measured concentrations:
- Nominal concentrations: 0.025, 0.083, 0.28, 0.91, 3.0 mg/L
- Mean Measured concentrations: 0.022, 0.073, 0.26, 0.78 and 2.3 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL glass bottle jars with teflon lined lids.
- Type: closed
- Material, size, headspace, fill volume: Glass bottle jars were completely filled with test solution to minimise headspace. Jars were covered with aluminium foil to reduce potential photodegradation.
- Aeration: No
- Renewal rate of test solution: Renewal after 24 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered well water (characterised as being moderately hard) from a well on the laboratory testing site.
- The water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered (0.45 µm) again to remove microorganisms and particles.
- Alkalinity: 176 to 180 mg/L (as CaCO3)
- Specific conductance: 310 to 315 µmhos/cm

OTHER TEST CONDITIONS
- 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 at the beginning and end of the 16-hour light period to avoid sudden changes in lighting.
- Light intensity: Light intensity measured at test initiation was approximately 288 lux over one representative test chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Observations were made to determine the number of mortalities and immobile organisms. Immobility was defined as the inability to swim within 15 seconds after gentle agitation of the test chamber. The number of individuals exhibiting clinical signs of toxicity or abnormal behaviour also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 0.43 and 0.79 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
MEASUREMENT OF TEST CONCENTRATIONS
- Two problems were encountered when measuring the test material concentration in the water. Firstly, in the 0.025 mg/L treatment group, 5 of the 8 samples collected were less than the limit of quantitation (LOQ = 0. 0200 mg/L). This occurred because the LOQ was so close to the nominal concentration. The mean measured concentration for this treatment is an estimate using the three samples which were >LOQ, and secondly the water samples for the 0.91 mg/L resulted in measured values that were 970 and 206 % of nominal. Analyses of the same solutions on Day 1 resulted in measured concentrations that were 83 and 86 % of nominal suggesting that the actual concentrations of the test solutions on Day 0 were not 970 and 206 % of nominal as measured. Based on the results of the Day 1 (old) samples, the measured concentrations on Day 0 must have been due to contamination during sampling or analysis. The Day 0 samples for this treatment group were not included in the calculation of the mean measured concentration.
- With the exceptions noted above, water samples collected at test initiation had measured concentrations that ranged from 70 to 94 % of nominal concentrations. "Old" samples collected after 24 hours of exposure had measured concentrations that ranged from 71 to 98 % of nominal. Consequently, the closed bottle system appeared to satisfactorily maintain the test concentrations. When measured concentrations of the samples collected throughout the test were averaged, the mean measured test concentrations for this study were 0.022, 0.073, 0.26, 0.78 and 2.3 mg/L, representing 88, 88, 93, 86 and 77 % of nominal concentrations, respectively. The results of the study were based on mean measured concentrations.

OBSERVATIONS AND MEASUREMENTS
- Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥ 8.8 mg/L (97 % of saturation) throughout the test. Measurements of pH ranged from 8.3 to 8.5 during the test. Measurements of hardness, alkalinity, total organic carbon (TOC) and specific conductance in the dilution water at test initiation were typical of the well water.
- Daily observations of mortality/immobility and other clinical signs of toxicity observed during the test are presented in Table 1. Daphnids in the negative control group appeared healthy and normal throughout the test. However, one daphnid in the negative control was not found on Day 1 of the test and was assumed to be dead. Daphnids in the 0.022 and 0.073 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity observed. After 48-hours of exposure, mortality/immobility in the 0.26, 0.78 and 2.3 mg/L treatment groups was 15, 55 and 100 %, respectively. EC50 values at 24 and 48 hours were estimated or calculated from the mortality data and are shown in Table 2.
Reported statistics and error estimates:
When the concentration-response pattern allowed for the calculation of an ECSO value, the data were analysed using a computer program. The program was designed to calculate the EC50 value and the 95 % confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. In this study, the moving average method was used to evaluate mortality/immobility at 48 hours because it provided the best fit to the concentration­response curve. The 24-hour EC50 value, the no-mortality/immobility concentration and NOEC were determined by visual interpretation of the mortality/immobility and biological observation data.

Table 1: Cumulative Percent Mortality and Observed Effects

Mean measured test concentration (mg/L)

Replicate

Daphnia/ replicate

4 hours

24 hours

48 hours

Percent immobile and dead (%)

Cumulative dead

Number immobile

Effects

Cumulative dead

Number immobile

Effects

Cumulative dead

Number immobile

Effects

Negative control

A

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

5

B

10

0

0

10 AN

1

0

9AN 1*

1

0

9 AN

0.022

A

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

0

B

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

0.073

A

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

0

B

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

0.26

A

10

0

0

10 AN

0

0

10 AN

1

2

7 AN

15

B

10

0

0

10 AN

0

0

10 AN

0

0

10 AN

0.78

A

10

0

0

10 AN

0

0

10 AN

0

8

2 AN

55

B

10

0

0

10 AN

0

0

10 AN

0

3

7 AN

2.3

A

10

0

0

10 AN

2

0

7AN 1C

10

0

-

100

B

10

0

0

10 AN

0

0

10 AN

10

0

-

AN= appear normal, C= lethargic and *= missing and assumed dead

Table 2: EC50 values

Time

EC50 (mg/L)

Lower 95 % Confidence limit (mg/L)

Upper 95 % Confidence limit (mg/L)

Statistical method

24    

>2.3

-*

-*

Visual inspection

48 hours

0.57

0.43

0.79

Moving average

-*= Confidence limits could not be calculated with the mortality data obtained

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the 48-hour EC50 value for Daphnia magna exposed to the test material was 0.57 mg/L. The 95 % confidence limits were 0.43 and 0. 79 mg/L. The 48-hour no mortality/immobility concentration and the NOEC was 0.073 mg/L. The 24-hour no mortality/immobility concentration and the NOEC was 0.78 mg/L.
Executive summary:

The potential acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EPA OPPTS 850.1010 under GLP conditions.

The toxicity of the test material was tested by treating Daphnia magna for a 48-hour exposure period under static-renewal test conditions. The daphnids were treated at the nominal concentrations of 0.025, 0.083, 0.28, 0.91, 3.0 mg/L. Test solutions were analysed using HPLC, the mean measured test concentrations for this study were 0.022, 0.073, 0.26, 0.78 and 2.3 mg/L, representing 88, 88, 93, 86 and 77 % of nominal concentrations, respectively. The results of the study were therefore based on mean measured concentrations.

Daily observations of mortality/immobility and other clinical signs of toxicity were recorded during the test. Daphnids in the negative control group appeared healthy and normal throughout the test. However, one daphnid in the negative control was not found on Day 1 of the test and was assumed to be dead. Daphnids in the 0.022 and 0.073 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity observed. After 48-hours of exposure, mortality/immobility in the 0.26, 0.78 and 2.3 mg/L treatment groups was 15, 55 and 100%, respectively. EC50 values at 24 and 48 hours were estimated or calculated from the mortality data.

Under the conditions of this study the 48-hour EC50 value for Daphnia magna exposed to the test material was 0.57 mg/L. The 95 % confidence limits were 0.43 and 0. 79 mg/L. The 48-hour no mortality/immobility concentration and the NOEC was 0.073 mg/L. The 24-hour no mortality/immobility concentration and the NOEC was 0.78 mg/L.

Description of key information

Under the conditions of this study the 48-hour EC50 value for Daphnia magna exposed to the test material was 0.57 mg/L. The 95 % confidence limits were 0.43 and 0. 79 mg/L. The 48-hour no mortality/immobility concentration and the NOEC was 0.073 mg/L. The 24-hour no mortality/immobility concentration and the NOEC was 0.78 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.57 mg/L

Additional information

The potential acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EPA OPPTS 850.1010 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The toxicity of the test material was tested by treating Daphnia magna for a 48-hour exposure period under static-renewal test conditions. The daphnids were treated at the nominal concentrations of 0.025, 0.083, 0.28, 0.91, 3.0 mg/L. Test solutions were analysed using HPLC, the mean measured test concentrations for this study were 0.022, 0.073, 0.26, 0.78 and 2.3 mg/L, representing 88, 88, 93, 86 and 77 % of nominal concentrations, respectively. The results of the study were therefore based on mean measured concentrations.

Daily observations of mortality/immobility and other clinical signs of toxicity were recorded during the test. Daphnids in the negative control group appeared healthy and normal throughout the test. However, one daphnid in the negative control was not found on Day 1 of the test and was assumed to be dead. Daphnids in the 0.022 and 0.073 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity observed. After 48-hours of exposure, mortality/immobility in the 0.26, 0.78 and 2.3 mg/L treatment groups was 15, 55 and 100%, respectively. EC50 values at 24 and 48 hours were estimated or calculated from the mortality data.

Under the conditions of this study the 48-hour EC50 value for Daphnia magna exposed to the test material was 0.57 mg/L. The 95 % confidence limits were 0.43 and 0. 79 mg/L. The 48-hour no mortality/immobility concentration and the NOEC was 0.073 mg/L. The 24-hour no mortality/immobility concentration and the NOEC was 0.78 mg/L.

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