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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:
2016-10-19 to
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Aliquots were removed from each prepared concentration every day for the range finding test and every day on fresh and old solutions for the definitive test.
Vehicle:
no
Details on test solutions:
For both range-finding test and definitive tests, as the test item was suspected to be unstable in water (due to its chemical nature), saturated stock solutions were prepared using slow-stirring conditions described in the OECD 123 guideline. The method used a special temperature controlled glass-jacketed test vessel (around 1L of capacity), with a sampling tap at the lower end and a magnetic stir bar at its bottom.

RANGE-FINDING TEST
A known volume of test medium was first poured into the flask then a known quantity of test item (1136.36 µL) was gently added at the surface of medium (corresponding to a loading rate of 1 g/L). The solution was stirred at 20°C with a magnetic stir bar at a speed avoiding formation of an emulsion. A speed of approximately 150 rpm was set up in order to create a vortex depth of around 0.5 cm. The aqueous phase was kept under stirring during approximately 29 hours, and then drawn off as a clear solution after a rest phase of a few minutes without agitation. Tests solutions were then prepared from dilutions of this saturated solution.

The pH of the test solutions were not adjusted.

Test solutions were not renewed throughout the 48h test period (static test).

DEFINITIVE TEST
As chemical analysis conducted during the preliminary test showed complete disappearance of the test item in test solutions throughout the 48h period, this definitive was conducted under semi-static conditions. An additional test item stock solution was prepared for test solutions renewal at 24h (semi-static test), as described in paragraph 3.4.1, except that the solutions (for the test initiation and the test solutions renewal) were prepared at 10 mg/L loading rate.

The pH of the test solutions were not adjusted.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test organisms used for this study was Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. Daphnia magna cultures were fed with a mixture of microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and did not belong to a first brood progeny.

Test animals were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperatures were maintained at 20 ± 1°C:

• Preliminary test: range from 20.5 to 20.6°C (mean 20.5°C)
• Definitive test: range from 20.5 to 20.6°C (mean 20.5°C)
pH:
RANGE-FINDING TEST:
0h: 7.3 - 8.0
48h: 7.4 - 7.6

DEFINITIVE TEST:
0h (fresh solution): 7.8 - 8.0
24h (old solution): 7.7 - 7.9
24h (fresh solution): 8.0
48h (old solution): 7.8 - 7.9
Dissolved oxygen:
RANGE-FINDING TEST:
0h: 8.4 - 8.7 mg/L
48h: 7.8 - 8.6 mg/L

DEFINITIVE TEST:
0h (fresh solution): 8.6 - 8.7 mg/L
24h (old solution): 8.4 - 8.6 mg/L
24h (fresh solution): 8.6 - 8.7 mg/L
48h (old solution): 8.4 - 8.6 mg/L
Nominal and measured concentrations:
RANGE-FINDING TEST:
Test solution (% saturated solution): 0 (Control), 6.25, 12.5, 25.0, 50.0, 100.0
Geometric means of measured concentrations: < Limit of Detection (Control), 0.04, 0.14, 0.27, 0.56, 1.18

DEFINITIVE TEST:
Test solution (% saturated stock solution): 0 (Control), 2.5, 5.2, 10.8, 22.7, 47.6, 100.0
Geometric means of measured concentrations: < Limit of Detection (Control), 0.01, 0.04, 0.08, 0.25, 0.52, 1.30
Details on test conditions:
Environmental Conditions
Culture and test vessels containing D. magna (20 daphnids per tested concentration for the definitive test) were incubated with a photoperiod of 8 hours of darkness per 24h at a temperature ranging from 18°C to 22°C but remaining constant within ± 1°C during the test.

Test vessels were made of clear glass tubes (around 20 mL capacity) and covered to minimise evaporation and avoid the entry of dust into the solutions. At least 3 mL of exposure solution was provided for each neonate.
 
The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility

Reference Item test

The sensitivity of the test system and the methodology areevaluated monthlyby performing an acute Daphnia magna toxicity test on potassium dichromate. The nearest value of 24h-EC50 obtained on October 2016 was 1.19 mg/L.

 

For information, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.

Environmental Conditions

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.

Validity criteria fulfilled:
yes
Conclusions:
This study was designed to determine the effects of the test item to Daphnia magna over a 48h period, according to the OECD 202 guideline. The range–finding test was performed under static conditions and the definitive under semi-static conditions.

Daphnids were exposed to serial dilutions of a saturated stock solution of the test item prepared under slow-stirring conditions due to the characteristics of the substance.

Chemical analysis of the test item throughout the test period have shown instability of the substance. Therefore, geometric means of measured concentrations were used to determine 48h-EC10 and 48h-EC50:

RANGE-FINDING TEST:
48-h EC10 = 0.41 mg/L (95% Confidence Interval: 0.20 - 0.57 mg/L)
48-h EC50 = 1.14 mg/L (95% Confidence Interval: 0.83 - 2.26 mg/L)
DEFINITIVE TEST:
48-h EC10 = 0.18 mg/L (95% Confidence Interval: Not determined due to mathematical reasons or inappropriate data)
48-h EC50 = 0.48 mg/L (95% Confidence Interval: Not determined due to mathematical reasons or inappropriate data)

Executive summary:

This study was designed to determine the effects of the test item to Daphnia magna over a 48h period, according to the OECD 202 guideline. The range–finding test was performed under static conditions and the definitive under semi-static conditions.

 

As the test item was suspected to be unstable in water (due to its chemical properties), tests solutions were prepared from dilutions of a saturated solution prepared using slow-stirring conditions described in the OECD 123 guideline.

 

Chemical analysis of the test item throughout the test period have shown instability of the substance. Therefore, geometric means of measured concentrations were used to determine 48h-EC10 and 48h-EC50 (all results are mg/L):

RANGE-FINDING TEST:

48-h EC10 = 0.41 mg/L (95% Confidence Interval: 0.20 - 0.57 mg/L)

48-h EC50 = 1.14 mg/L (95% Confidence Interval: 0.83 - 2.26 mg/L)

DEFINITIVE TEST:

48-h EC10 = 0.18 mg/L (95% Confidence Interval: Not determined due to mathematical reasons or inappropriate data)

48-h EC50 = 0.48 mg/L (95% Confidence Interval: Not determined due to mathematical reasons or inappropriate data)

The definitive test met the validity criteria of the test guideline detailed as follows:

  • The immobilisation in the control did not exceed 10% at the end of the test (0% in the definitive test),
  • The dissolved oxygen concentrations remained above 3 mg/L over the test period (above 8.4 mg/L in the definitive test).

Description of key information

The 48h-EC50 determined from data generated by a test performed according to OECD Test Guideline 202 was 0.48 mg/L for Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.48 mg/L

Additional information

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