Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Due to the required volume for dissolved copper analyses, additional flasks (two per concentration with daphnids) were included in the test protocol to perform analytical measurements at intermediate time of 24 hours.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is incuded in the report

Analytical monitoring:

yes

Details on sampling:

Analyses were performed on test solutions at T0h, T24h and T48h. Two additional vessels per concentration (with daphnids) were used for analysis for the sampling time 24 hours.

Vehicle:

no

Details on test solutions:

Available information has shown that the test item is not soluble in water. A stock solution was prepared in the dilution water (nominal concentration: 500.0 mg/L), in the dark under magnetic stirring, one week before initiating the Daphnia magna acute toxicity test. After filtration (PTFE syringe filter 0.45 µm pore size), the stock solution was diluted in the dilution water to achieve the required test concentrations. Most of the copper was undissolved and was removed by filtration.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus 1820 (Crustacea Cladocera) coming from a continuous and balanced breeding carried out in the test facility, aged less than 24 hours at the start of the test.Culture conditions: Medium: Elendt M4Food: TetraMin + green algaeLight: 2500 lux +/- 400 lux ; 16-hours light / 8-hours dark cycleTemperature: 20 ± 2°C

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Dilution water (reconstituted water – NF EN ISO 6341): 239.4 mg/L expressed as CaCO3

Test temperature:

between 19.5 and 20.4°C

pH:

Control: between 7.7 and 7.8Test solutions: 7.8 and 8.7

Dissolved oxygen:

Control: between 8.7 and 8.8 mg O2/LTest solutions: 8.5 and 8.9 mg O2/L

Salinity:

Not available

Conductivity:

Dilution water (reconstituted water – NF EN ISO 6341): 648 µS/cmStock solution (500 mg/L) after filtration to 0.45 µm: 845 µS/cm

Nominal and measured concentrations:

100.0 – 149.5 - 223.5 - 334.5 – 500.0 mg/L (CuDABT initial nominal concentrations)

Details on test conditions:

Vessels: disposable glass tubes filled with 10 ml of test solution.Number of animals: 30 daphnids divided in six groups of five animals, for each test solution and for the control. Test concentrations: 100.0 – 149.5 - 223.5 - 334.5 – 500.0 mg/L (initial nominal concentrations)Loading: 2 mL of test solution for each daphnidFeeding: none within the test periodAeration: none within the test periodEnvironmental conditions: complete darkness; temperature between 19.5 and 20.4°CRenewal of test solution: noneTime of observation: 24 h and 48 hToxic effect: immobilization (Daphnids that are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobilised (even if they can still move their antennae))

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 334.5 - < 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: No mortality to 334.5 mg/l and 100% mortality to 500.0 mg/L.

Remarks:

Nominal concentration equivalent respectively to 113 mg/L to 5,5’-bi-1H-tetrazole (diammonium salt) and 8.6 µg/L to dissolved copper for 334.5 mg/L and 178 mg/L to 5,5’-bi-1H-tetrazole (diammonium salt) and 13.7 µg/L to dissolved copper for 500.0 mg/L.

Details on results:

Stability of samples has been demonstrated during the validation study regarding the analysis of DABT (study 16-003-156922; report DRC 16-156922-07470A).
Samples were stored to 4°C until analysis.

Results with reference substance (positive control):

The test carried out from November 23rd to 24th 2016 gave the following results: EC50 24h: 1.07 mg/L (1.03 – 1.16 mg/L).The EC50 24 hours was calculated using a logistic regression analysis with a bootstrap estimation of the 95% intervals.The EC50 24h is in the recommended range: 0.6 – 2.1 mg/L. This result confirms the appropriate sensitivity of the strain of Daphnia magna

Definitive test

Results obtained for the four vessels per test concentrations are presented in tables below:

 Observation at 24h

 

Nominal test concentrations (mg/L)	Filtered stock solution (%)	Number of daphnids immobilised per test vessel					% of immobilization
		A	B	C	D	Total (/20)
Controls	0	0	0	0	0	0	0
100.0	20.0	0	0	0	0	0	0
149.5	29.9	0	0	0	0	0	0
223.5	44.7	0	0	0	0	0	0
334.5	66.9	0	0	0	0	0	0
500.0	100	5	5	5	5	20	100

  Observation at 48h

 

Nominal test concentrations (mg/L)	Filtered stock solution (%)	Number of daphnids immobilised per test vessel					% of immobilization	pH	O2(mg/L)
		A	B	C	D	Total (/20)
Controls	0	0	0	0	0	0	0	7.7	8.7
100.0	20.0	0	0	0	0	0	0	7.8	8.8
149.5	29.9	0	0	0	0	0	0	7.8	8.8
223.5	44.7	0	0	0	0	0	0	7.8	8.8
334.5	66.9	0	0	0	0	0	0	7.9	8.8
500.0	100	5	5	5	5	20	100	8.1	8.9

Validity criteria fulfilled:

yes

Remarks:

In the control vessels, the immobilization of the daphnids did not exceed 10% at the end of the test. The dissolved oxygen concentration at the end of the test was > 3 mg/L in control and in test concentrations.

Conclusions:

In the test conditions, EC50 48h of the test item CuDABT has been determined between 334.5 and 500.0 mg/L (initial nominal concentrations).
EC50 > 334.5 mg/L.

Executive summary:

Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test item CuBABT at a range of concentrations between 100.0 and 500.0 mg/L (initial nominal concentrations) for a period of 48 hours.

Immobilization is recorded at 24 hours and 48 hours.

In the test conditions, EC₅₀ 48h of the test item CuDABT have been determinate between 334.5 and 500.0 mg/L (initial nominal concentrations).

As mortamity was 100 % at 500.0 mg/L, EC50 CuDABT 48h > 334.5 mg/L

Description of key information

In the test conditions, EC50 48h of the test item CuDABT has been determined between 334.5 and 500.0 mg/L (initial nominal concentrations).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

334.5 mg/L