Copper diformate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-02 to 2020-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

No. 440/2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper diformate

EC Number:

208-865-8

EC Name:

Copper diformate

Cas Number:

544-19-4

Molecular formula:

CH2O2.1/2Cu

IUPAC Name:

copper diformate

Test material form:

solid: particulate/powder

Details on test material:

blue in colour

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During final test:
- Concentrations: control and all test concentration
- Sampling method: fresh samples from the bulk test preparation at 0 and 24 hours/ aged samples from pooled replicates at 24 and 48 hours
- Sample storage conditions before analysis: freshly prepared and aged sample of 24 hours were immediately analysed/ aged 48 hours samples were initially frozen prior to analysis

Test solutions

Vehicle:

no

Details on test solutions:

A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication for 1 minute and the volume adjusted to 500 mL to give a 100 mg/L stock concentration. This was diluted to make a 10 mg/L stock concentration from which a series of dilutions was made to give the test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
The stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation/Stage and instar at study initiation: first instar daphnids < 24 hours
- Source: in-house laboratory cultures
- Method of breeding (Culture conditions) : reproduction of adult daphnids was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Holding conditions (Culture): temperature: 18-22 °C; light: 16 hours a day; feeding: daily with a mixture of algae and GEMMA Micro 300 fish food suspension
- Feeding during test: no

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L CaCo3

Test temperature:

20 to 21°C

pH:

7.9 to 8.1

Dissolved oxygen:

8.7 to 9.1

Salinity:

not applicable

Nominal and measured concentrations:

- Nominal: 0.010, 0.032, 0.10, 0.32 and 1.0 mg test item/L
- Geometric mean measured test concentrations for copper: 0.0053, 0.080, 0.020, 0.089 and 0.31 mg copper/L
- Geometric mean measured test concentrations for copper diformate: 0.013, 0.020, 0.048, 0.21 and 0.74 mg copper diformate/L

Details on test conditions:

Test System
- Test vessel: glass ware, size: 240 mL beakers containing 100 mL of test solution or control media
- Aeration: none
- Medium: reconstituted water (ISO-medium)
- No. of organisms/vessel: 5
- Replicates: 4 per test concentration and control
- photoperiod: 16 h light: 8 h darkness (with 20 minute dawn and dusk transition periods)
- one water renewal after 24 hours of exposure
- pH values: 0/24 h, fresh media: 7.9-8.1, 24/48 h, aged media: 7.9-8.1
- Oxygen value: 0/24 h, fresh media: 8.8-9.0 mg/L, 24/48 h, aged media: 8.7-9.1
- Temperature: 18-22 +-1 °C

Test concentrations:
- initial range finding test: 0.10, 1.0, 10 and 100 mg/L -> toxicity at all test concentrations
- second range finding test: 0.0010, 0.0050, 0.010 and 0.10 mg/L
- final test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

effect concentration based on copper diformate

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

effect concentration based on copper diformate

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

effect concentration based on copper diformate

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits 0.079 - 0.15 mg/L

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

effect concentration based on copper diformate

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.089 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

mobility

Effect concentrations 7

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits 0.033 - 0.063 mg/L

Effect concentrations 8

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.078 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

mobility

Details on results:

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Temperature was maintained at 20 to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 847 to 1034 Lux.
No sub-lethal effects of exposure were observed throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50(48h): 0.71 mg/L (95% CI: 0.61 - 0.81 mg/L)
- Other: The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) were calculated using the Step-down Cochran-Armitage test. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Any other information on results incl. tables

Range finding test:

Immobilization was observed at all test concentrations in the initial range-finding test, 70% immobilization at the test concentration of 0.10 mg/L and 100% immobilization at test concentrations of 1.0, 10 and 100 mg/L.

In the second range-finding test, no immobilization was observed at test concentrations of 0.0010, 0.0050 and 0.010 mg/L, however; 30% immobilization was observed at 0.10 mg/L.

Reduced mobility was observed at a test concentration of 0.10 mg/L.

Based on this information nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

Chemical analysis of the test preparations from the second range-finding test showed measured test concentrations of copper to range from 93 to 525% of the nominal concentration at 0 hours and 64 to 394% of the nominal concentration at 48 hours. High values obtained at lower concentrations were considered to be due to proximity to the limit of quantification (LOQ) for the method of analysis employed.

Main Test:

Verification of Test Concentrations - copper:

ICP analysis for copper of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.011 to 0.38 mg/L and of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0078 mg/L, to 0.38 mg/L at 24 hours. ICP analysis for copper of the corresponding aged test preparations showed measured test concentrations of less than the LOQ to 0.25 mg/L at 24 hours and of less than the LOQ to 0.25 mg/L at 48 hours.

For the control and all test groups at 0 hours, duplicate samples which had been stored frozen were provided for analysis due to the detection of trace levels of copper in the control and achieved values outside of the acceptable 80-120% of nominal range. As the duplicate values were not significantly different to the initial values, the mean values were used for calculation and reporting purposes.

It was considered that it was appropriate to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The average geometric mean measured test concentrations were determined to be 0.0053, 0.080, 0.020, 0.089 and 0.31 mg/L for copper.

Immobilization - copper:

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.9 (standard error = 0.73) and 4.5 (standard error = 0.066) respectively.

Verification of Test Concentrations - copper diformate:

ICP analysis for Copper Diformate of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.027 to 0.92 mg/L and of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.019 mg/L, to 0.91 mg/L at 24 hours. ICP analysis for Copper Diformate of the corresponding aged test preparations showed measured test concentrations of less than the LOQ to 0.61 mg/L at 24 hours and of less than the LOQ to 0.60 mg/L at 48 hours.

For the control and all test groups at 0 hours, duplicate samples which had been stored frozen were provided for analysis due to the detection of trace levels of Copper Diformate in the control and achieved values outside of the acceptable 80-120% of nominal range. As the duplicate values were not significantly different to the initial values, the mean values were used for calculation and reporting purposes.

It was considered that it was appropriate to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The average geometric mean measured test concentrations were determined to be 0.013, 0.020, 0.048, 0.21 and 0.75 mg/L for Copper Diformate.

Immobilization - copper diformate:

The slopes and their standard errors of the response curves at 24 and 48 hours were 9.0 (standard error = 0.71) and 4.6 (standard error = 0.065) respectively.

Table 1: Cumulative immobilization data after 24 and 48 h in the definitive test

Nominal test item Concentration [mg/L]	24 h
	Cumulative ImmobilizedDaphnia(Initial Population: 5 daphnids per replicate)
	R1	R2	R3	R4	Total	%
24 h
Control	0	0	0	0	0	0
0.010	0	0	0	0	0	0
0.032	0	0	0	0	0	0
0.10	0	0	0	0	0	0
0.32	0	4	5	5	14	70
1.0	5	5	5	5	20	100
48 h
Control	0	0	0	0	0	0
0.010	0	0	0	0	0	0
0.032	0	0	0	0	0	0
0.10	1	0	0	0	1	5
0.32	3	5	5	5	18	90
1.0	5	5	5	5	20	100

Table 2: Analytical results of test sample measurements

Time point	Nominal concentration of test item in test sample	Determined concentration if copper in test sample	Determined concentration if test item in test sample	Percentage of nominal concentration
[hours]	[mg/L]	[mg Cu/L]	[mg test item/L]	[%]
0 h (test start)	Control	0.0105 (< LOQ)	0.0253 (< LOQ)	-
	0.01	0.0135	0.0327	327
	0.032	0.0187	0.0452	141
	0.1	0.0424	0.102	102
	0.32	0.128	0.310	97
	1.0	0.382	0.922	92
P. Rec 1	0.100	0.108	-	108
P. Rec 2	0.100	0.108	-	108
24 h (aged)	Control	< LOQ	< LOQ	-
	0.01	< LOQ	< LOQ	-
	0.032	< LOQ	< LOQ	-
	0.1	0.0116	0.0279	28
	0.32	0.0619	0.150	47
	1.0	0.251	0.607	61
P. Rec 1	0.100	0.103	-	103
P. Rec 2	0.100	0.102	-	102
24 h (fresh)	Control	< LOQ	< LOQ	-
	0.01	< LOQ	< LOQ	-
	0.032	0.0139	0.0336	105
	0.1	960.385	0.0930	93
	0.32	0.127	0.306	96
	1.0	0.378	0.914	91
48 h (aged)	Control	< LOQ	< LOQ	-
	0.01	< LOQ	< LOQ	-
	0.032	< LOQ	< LOQ	-
	0.1	0.00836	0.0202	20
	0.32	0.0624	0.151	47
	1.0	0.249	0.601	60
P. Rec 1	0.100	0.0985	-	99
P. Rec 2	0.100	0.102	-	102

Note: The concentrations quoted for the procedural recovery samples are the actual fortified concentrations of copper. The corresponding nominal concentration values relate to the percentage of fortified concentration recovered.

P. rec = procedural recovery; LOQ = limit of quantification (0.0078 mg/L for copper / 0.019 mg/L for test item); - = not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:
Based on copper: EC50 48h: 0.046 mg/L
Based on copper diformate: EC50 48h: 0.11 mg/L