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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted April 13, 2004
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch number: CN61204501
Date of receipt: 2017-03-02
Expiry date: 2018-07-24
Stability of test concentration/s during exposure: Examined by chemical analysis (Zinc) in the freshly prepared media and in the media after 24 hours of exposure according to the semi-static test conditions.

Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and preparation of test item concentrations
A stock solution was prepared to give the desired series of test concentrations. 101 mg of the test item were added to 1 litre of dilution water on 2018-03-05 and 99.9 mg of the test item were added to 1 litre of dilution water on 2018-03-06, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of
7 - 12 µm. The pH was measured to be 7.9 on 2018-03-06 and 7.8 on 2018-03-07.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 500 mL. 50 mL of the prepared solutions were taken per replicate and 0.5 mL of dilution water containing 5 daphnids was given to all replicates resulting in the final nominal concentrations. For each test item concentration and the control 4 replicates were prepared.


Test organisms (species):
Daphnia magna
Details on test organisms:
Name: Daphnia magna STRAUS, parthenogenetic females
Source: Strain of Bundesgesundheitsamt Berlin
Maintenance and Acclimatisation: A population of parthenogenetic females of synchronized age structure has been maintained for more than 20 years in the test facility under constant temperature conditions (20 +/- 2 °C) at a 16 : 8 hour light-dark photoperiod (light intensity: < 20 µE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily. The neonates were separated from their parent Daphnia by filtration prior to the acute test.

Culture and dilution water: Reconstituted water (so-called 'M4 medium' according to OECD 202 and EC Method C.2, annex 1) was used for the maintenance of the test animals and the preparation of stock and test solutions of the test item.

Test type:
semi-static
Water media type:
freshwater
Remarks:
The total hardness of the dilution water, measured at test start, was 14.5 °dH (= 259 mg/L CaCO3).
Limit test:
no
Total exposure duration:
48 h
Hardness:
14.5 °dH (= 259 mg/L CaCO3)
Test temperature:
18 - 22 °C +/- 1 °C
pH:
7.9 - 8.5
Dissolved oxygen:
8.3 - 8.7 mg/L (97 - 101 %)
Nominal and measured concentrations:
nominal: 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, 31.6 and 100 mg/L
Details on test conditions:
EXPOSURE CONDITIONS
Test vessels: 100 mL glass beakers covered with watch glasses holding 5 neonates in 50 mL of test medium
Experimental design: 9 test concentrations plus 1 control; 5 neonates per vessel, 4 replicates per concentration/control; no feeding during the exposure period; semi-static system
Method of initiation: neonates were placed in prepared media
Photoperiod: 16 h light : 8 h dark
Temperature of incubation unit: 19.2 to 19.8 °C
Aeration: none
Test item concentration/s: 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, 31.6 and 100 mg/L
Medium renewal: daily
Duration of exposure: 48 hours
Criteria of effects: The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
138.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 675.5 - 1322.5 µg/L
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
4 292.9 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 675.5 - 1322.5 µg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
947 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 675.5 - 1322.5 µg/L
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
6.3 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 31.5 - 75.4 µg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1 471.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 31.5 - 75.4 µg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
48.7 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 31.5 - 75.4 µg/L
Details on results:
The results are expressed in terms of arithmetic mean measured. Concentrations (at 24 and 48 h). The results refer to the test item concentrations calculated on base of the measured Zinc values. According to the product information 1 mg/L Zinc equals 7.01 mg/L test item (molecular weight 458 g/mol, empirical formula C18H20N2O4 Zn).
Effective concentrations ranged from 4.4 % to 14.2 % of nominal values at 0 hours, and from 4.1 % to 4.7 % of nominal values at 48 hours.
With the sponsor’s agreement, the content of the test item during the exposure period was verified by Zn-determination.
Reported statistics and error estimates:
The EC 50 was calculated by probit analysis using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

Results of the probit analysis

Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95-confidence limits (according to Fieller`s theorem).

24h

 Parameter  EC10  EC20  EC50
 Value [µg/L]  336.510  480.008  946.983
 lower 95%-cl  166.716  278.788  675.527
 upper 95%-cl  498.018  673.207  1322.500

Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%-confidence limits (according to Fieller`s theorem).

48h

Parameter  EC10  EC20  EC50
 Value [µg/L]  6.671  13.195  48.653
 lower 95%-cl  2.912  6.902  31.538
 upper 95%-cl  11.603  21.086  75.428
Validity criteria fulfilled:
yes
Conclusions:
A study was performed to assess the acute toxicity of Vulkacit P Extra N to Daphnia magna STRAUS under semi-static conditions. An EC50 value of 48.7 µg/L was determined according to Council Regulation (EC) No 440/2008, Method C.2.
Executive summary:

A study was performed to assess the acute toxicity of Vulkacit P Extra N to Daphnia magna STRAUS under semi-static conditions.

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004).

The Daphnia were exposed to a range of concentrations, nominally 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, 31.6 and 100 mg/L of Vulkacit P Extra N dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and folded filters.

Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure. The following values were determined:

 Time [h]  EC50 [µg/L]
 24  947.0
 48  48.7

The results are expressed in terms of arithmetic mean measured concentrations (at 24 and 48 h). The results refer to the test item concentrations calculated on base of the measured Zinc values. According to the product information of the sponsor 1 mg/L Zinc equals to 7.01 mg/L test item (molecular weight 458 g/mol, empirical formula C18H20N2O4 Zn).

Effective concentrations ranged from 4.4 % to 14.2 % of nominal values at 0 hours, and from 4.1 % to 4.7 % of nominal values at 48 hours.

With the sponsor’s agreement, the content of the test item during the exposure period was verified by Zinc-determination.

The hardness of the dilution water used was 14.5 °dH (= 259 mg/L CaCO3).

Description of key information

A study was performed to assess the acute toxicity of Vulkacit P Extra N to Daphnia magna STRAUS under semi-static conditions.

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004).

The Daphnia were exposed to a range of concentrations, nominally 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, 31.6 and 100 mg/L of Vulkacit P Extra N dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and folded filters.

Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure. The following values were determined:

 Time [h]  EC50 [µg/L]
 24  947.0
 48  48.7

The results are expressed in terms of arithmetic mean measured concentrations (at 24 and 48 h). The results refer to the test item concentrations calculated on base of the measured Zinc values. According to the product information of the sponsor 1 mg/L Zinc equals to 7.01 mg/L test item (molecular weight 458 g/mol, empirical formula C18H20N2O4 Zn).

Effective concentrations ranged from 4.4 % to 14.2 % of nominal values at 0 hours, and from 4.1 % to 4.7 % of nominal values at 48 hours.

With the sponsor’s agreement, the content of the test item during the exposure period was verified by Zinc-determination.

The hardness of the dilution water used was 14.5 °dH (= 259 mg/L CaCO3).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
48.7 µg/L

Additional information