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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:
11 - 13 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliche Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany (03.01.2017)
Test material information:
Composition 1
Analytical monitoring:
yes
Remarks:
HPLC-DAD
Details on sampling:
- Concentrations: Control, 0.625, 1.25, 2.50, 5.00, and 10.0 mg/L
- Sampling method: Samples of the freshly prepared test solutions were taken at the start of exposure and at medium renewal (0 and 24 h). Samples of aged, 24-h old test solutions, were taken directly from the test vessels at renewal and at the end of exposure (24 and 48 h).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
5 test item concentrations in a geometric series with a separation factor of 2 were prepared by dilution of a stock solution of 10.0 mg/L with dilution water. The stock solution was mixed thoroughly by manual agitation.
- Controls:
Dilution water without test item and without solvent incubated under the same conditions as the test groups.
- Evidence of undissolved material: All tested concentration levels were visually clear at the start of the exposure and at renewal after 24 h. The old media after 24 and 48 h were slightly brown at the two highest concentration levels. All other concentration levels were clear and colorless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Age of daphnids at test start: < 24 h old. Juvenile daphnids were removed from the culture vessels the latest 24 h before the start of exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Culture: In 2 - 3 L glass vessels with approximately 1.8 L culture medium at 20 ± 2 °C, in an incubator with 16 h illumination (max. 1500 lx)
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), with total hardness modified to 160 - 180 mg CaCO3/L
- Culture feeding: 5 times per week ad libitum with a mix of unicellular green algae (P. subcapitata and D. subspicatus, density > 10^6 cells/mL)
- Feeding during test: No

ACCLIMATION
Not necessary because the dilution water was equivalent to the culture medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
164 - 166 mg CaCO3/L
Test temperature:
20.1 - 20.6 °C
pH:
7.18 - 7.92 (control)
7.31 - 8.02 (treatments)
Dissolved oxygen:
8.62 - 9.48 mg O2/L (control)
8.54 - 9.13 mg O2/L (treatments)
Conductivity:
414 - 419 µS/cm
Nominal and measured concentrations:
Control, 0.625, 1.25, 2.50, 5.00, and 10.0 mg/L (nominal)
< LOQ , 0.613, 1.18, 2.32, 3.19, and 4.83 mg/L (geom. mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers (4 (ID) x 7 (H) cm), loosely covered with watch glasses
- Fill volume: 20 mL
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water was same as culture medium.
- Conductivity: 414 - 419 µS/cm
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: In fresh media, the water quality parameters were measured at the start of exposure and at the renewal of water (0 and 24 h) in one additional replicate (without daphnids) per concentration level and control. In aged media, the water quality parameters were meausred at renewal and at the end of exposure (24 and 48 h) in one replicate (containing daphnids) per concentration level and control.

OTHER TEST CONDITIONS
- Photoperiod:16 h light, 8 h dark cycle
- Light intensity: max. 1500 lx

EFFECT PARAMETERS MEASURED:
- Immobilization: after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: Control, 1, 10, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: 100% immobilization after 48 h of exposure to 10 mg/L test item. After 48 h the test item concentrations had decreased by 16 - 72%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 2.32 - 4.83 mg/L
Details on results:
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: All tested concentration levels were visually clear at the start of the exposure and at the renewal after 24 h. The old test media after 24 and 48 h were slightly brown at the two highest concentration levels. All other concentration levels were clear and colorless.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid?
Yes. A reference test with a test duration of 24 h is conducted once per month at the test facility. The most recent test dates from 11 - 12 Jul 2017 and produced results within the valid range of 0.6 - 2.1 mg/L, as defined by the OECD guideline 202.
- EC50 (24 h): 1.31 mg/L
, 95% confidence limits: 1.05 - 1.72 mg/L
Reported statistics and error estimates:
The EC10 and EC100 values were empiriacally determined from data. The EC50 values were calculated by sigmoidal dose-response regression using GraphPadPrism.
FITTING RESULTS OF THE ESTIMATION OF THE EC10 AND EC50 VALUE AFTER 48 HOURS
Equation: sidmoidal dose-response, variable slope
y = bottom + (top-bottom)/(1+10^((logEC50 - x)*HillSlope))
Transform: x = log (x)
BEST-FIT VALUES
Bottom: -8.7E-009; Top: 100.0; log EC50: 0.4937; HillSlope: 74.74; EC10: 3.027; EC50: 3.117; EC100: 3.864
GOODNESS OF FIT
Degress of Freedom: 16; R square: 0.9928; Absolute sum of squares: 300.0; Sy.x: 4.330

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the OECD guideline 202 (Table 1).

Table 1: Validity criteria OECD guideline 202.

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

No immobilization or any signs of distress were recorded in the control.

Yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

The dissolved O2 concentration in the 24-h old media at the end of the exposure intervals was ≥ 8.54 mg/L in all tested concentration levels and the control.

Yes

ANALYTICAL RESULTS

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 h) ranged from 70 – 107% of the nominal values, and 28 – 101% in old media at renewal and at the end of the test (24 and 48 h) (Table 2). Since the measured concentrations were not within ± 20% of nominal concentrations, effect concentrations were based on calculated geometric mean measured concentrations.

 

Table 2. Measured concentrations and percent of nominal concentration of the test item.

Sampling time

0 hours

fresh media

24 hours

fresh media

24 hours

old media

48 hours

old media

Geometric mean measured test item concentrations

[mg/L]

Nominal test item concentration

[mg/L]

meas. conc.

[mg/L]

%

meas. conc.

[mg/L]

%

meas. conc.

[mg/L]

%

meas. conc.

[mg/L]

%

10.0

8.25

83

2.83

28

n.d.

n.d.

n.d.

n.d.

4.83

5.00

3.48

70

1.98

40

3.89

78

3.86

77

3.19

2.50

2.54

102

1.75

70

2.59

104

2.53

101

2.32

1.25

1.27

102

0.969

78

1.29

103

1.24

99

1.18

0.625

0.668

107

0.524

84

0.647

104

0.624

100

0.613

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. conc.      = measured concentration of the test item

%                         = percent of the nominal concentration of the test item

LOQ                     = limit of quantification of the analytical method (0.0625 mg/L test item)

n.d.                      = not determined

  

BIOLOGICAL RESULTS

Immobilization was observed in the two highest treatment levels after 24 and 48 h exposure (Table 3).

The derived EC50 (48 h) was 3.17 mg/L (geometric mean measured) with a 95% confidence interval of 2.32 - 3.19 mg/L and the EC10 (48 h) was reported as 2.32 mg/L < EC10 < 3.19 mg/L (geometric mean measured).

Table 3. Immobilization Rates after 24 and 48 hours of exposure in the definitive test (n = 20, divided into 4 replicates with 5 daphnids each)

 

 

Nominal test item concentration

[mg/L]

Geometric mean measured test item concentration

[mg/L]

Immobilization [%]

24 hours

48 hours

replicates

replicates

1

2

3

4

MV

1

2

3

4

MV

10.0

4.83

100

100

100

100

100

100

100

100

100

100

5.00

3.19

60

80

60

40

60

80

100

80

80

85

2.50

2.32

0

0

0

0

0

0

0

0

0

0

1.25

1.18

0

0

0

0

0

0

0

0

0

0

0.625

0.613

0

0

0

0

0

0

0

0

0

0

Control

 

0

0

0

0

0

0

0

0

0

0

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The experimentally determined EC50 (48 h) for the short-term toxicity to aquatic invertebrates is 3.12 mg/L (geometric mean measured concentration, OECD 202, D. magna).

Description of key information

EC50 (48 h) = 3.12 mg/L (geom. mean measured, OECD 202, D. magna)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
3.12 mg/L

Additional information

There is one study available, in which the short-term toxicity of 2-Hydroxycyclohepta-2,4,6-trienone (CAS 533-75-5) to aquatic invertebrates was assessed according to OECD guideline 202 and GLP.

In a semi-static test with a 24 h renewal rate, Daphnia magna was exposed to 5 concentrations in a geometric series ranging from 0.625 – 10.0 mg/L test item (nominal) and a separation factor of 2 for 48 h. Test item concentrations were analytically verified by HPLC-DAD in fresh media (0 and 24 h) and in aged media (24 and 48 h).

The measured concentrations of the test item in fresh media ranged from 70 – 107% of the nominal values and from 28 – 104% of nominal in aged media. Therefore, effect concentrations are expressed as geometric mean measured values. The test resulted in an EC50 (48 h) of 3.12 mg/L.