Copper diformate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-21 to 2020-03-02

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.3 (Algal Inhibition test)

Version / remarks:

761/2009

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

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:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
An additional sample of each test concentration was incubated alongside to provide samples at 24 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

ultrasonication for approximately 1 minute and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 0.010, 0.032, 0.10, 0.32, 1.0 and 10 mg/L. An aliquot (900 mL) of each of the 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L stock solutions was separately inoculated with algal suspension (6.3 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The solutions were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.
The culture medium used for both the range-finding and initial test was the same as that used to maintain the stock culture. The culture medium used for the definitive test was modified by the addition of 198.4 mg of CaCl2.2H2O/L in order to help avoid the complexing effects of the test item and prevent a complexation-induced nutrient limitation which could have impaired algal growth.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ±1 ºC

pH:

0 hours: 7.6 to 7.8
72 hours: 7.9 to 8.1

Nominal and measured concentrations:

- Nominal: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
- geometric mean measured as copper diformate: 0.0080, 0.038, 0.092, 0.27 and 0.90 mg/L
- geometric mean measured as copper: 0.0033, 0.016, 0.038, 0.011 and 0.37 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks containing 100 mL of test media
- Type: plugged with polyurethane foam bungs to reduce evaporation
- Aeration: none
- Initial cells density: 5.00 x 10^3 cells/mL
- Control end cells density: 8.37 x 10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (ASTM media)

TEST MEDIUM / WATER PARAMETERS
- Test medium different from culture medium: culture medium was modified by the addition of 198.4 mg of CaCl2x2H2O/L in order to help avoid the complexing effects of the test item and prevent a complexation-induced nutrient limitation which could have impaired algal growth
- Intervals of water quality measurement: pH values were measured at test start and test end of the control and each test preparation. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)
- others: test vessels were constantly shaken at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter® Multisizer Particle Counter)/ Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
- Other: To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1
- Initial Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- 2nd range finding study
- Test concentrations: 0.010, 0.10 and 1.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 0.18 to 0.23 mg/L

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.054 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.083 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 0.072 to 0.096 mg/L

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

growth rate

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

biomass

Effect concentrations 12

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

biomass

Effect concentrations 13

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% confidence limits 0.041 to 0.052 mg/L

Effect concentrations 14

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.016 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

biomass

Effect concentrations 15

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

copper

Basis for effect:

biomass

Effect concentrations 16

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.064 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

biomass

Effect concentrations 17

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.079 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

biomass

Effect concentrations 18

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 0.10 to 0.13 mg/L

Effect concentrations 19

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

biomass

Effect concentrations 20

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

copper diformate

Basis for effect:

biomass

Details on results:

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.010, 0.032 and 0.10 mg/L, however no intact cells were observed to be present in the test cultures at 0.32 and 1.0 mg/L.

Water Quality Criteria:
Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility:
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.010, 0.032 and 0.10 mg/L test cultures were green or slightly pale green dispersions whilst the 0.32 and 1.0 mg/L test cultures remained as clear colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 1.3 mg/L; 95% confidence limits 1.2 to 1.4 mg/L
- EyC50: 0.55 mg/L; 95% confidence limits 0.49 to 0.63 mg/L

Reported statistics and error estimates:

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Range-finding Tests:

The results showed no effect on growth at a test concentration of 0.10 mg/L, however growth was observed to be reduced at concentrations of 1.0, 10 and 100 mg/L.

The results showed a significant effect on yield at test concentrations of 0.10, 1.0, 10 and 100 mg/L in the initial range-finding test.

The results of the second range-finding test showed no effect on growth at test concentrations of 0.010 and 0.10 mg/L, however growth was observed to be reduced at a concentration of 1.0 mg/L.

The results of the second range-finding test showed no effect on yield at a test concentration of 0.010 mg/L. However, yield was observed to be reduced at 0.10 and 1.0 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.

Main Test:

Verification of test concentration - copper:

ICP-MS analysis for copper of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0066 mg/L for the method of analysis utilized in this study, to 0.42 mg/L and less than the LOQ to 0.33 mg/L at 72 hours.

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 0.0033, 0.016, 0.038, 0.011 and 0.37 mg/L as copper.

Growth data - copper:

From the data obtained, it is clear that the growth rate and yield of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

There were no statistically significant differences (P=0.05), between the control and 0.0033, 0.016 and 0.038 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.038 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.11 mg/L.

Inhibition of yield - copper:

There were no statistically significant differences (P=0.05), between the control and 0.0033 and 0.016 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 0.016 mg/L. Correspondingly the LOEC based on yield was 0.038 mg/L.

Verification of test concentration - copper diformate:

ICP-MS analysis for Copper Diformate of the test preparations at 0 hours showed measured test concentrations to range from less than the LOQ, determined to be 0.016 mg/L for the method of analysis utilized in this study, to 1.0 mg/L and less than the LOQ to 0.81 mg/L at 72 hours.

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 0.0080, 0.038, 0.092, 0.27 and 0.90 mg/L as Copper Diformate.

There were no statistically significant differences (P=0.05), between the control and 0.0080, 0.038 and 0.092 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.092 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.27 mg/L.

Growth data - copper diformate:

From the data obtained, it is clear that the growth rate and yield of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Inhibition of yield - copper diformate:

There were no statistically significant differences (P=0.05), between the control and 0.0080 and 0.038 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 0.038 mg/L. Correspondingly the LOEC based on yield was 0.092 mg/L.

Table 1: Inhibition of growth rate and yield based on cell density after 72 h of exposure to copper diformate (definitive test)

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.071	-	8.07E+05	-
	R2	0.070		7.85E+05
	R3	0.071		8.18E+05
	R4	0.073		9.20E+05
	R5	0.071		8.29E+05
	R6	0.071		8.31E+05
	Mean	0.071		8.32E+05
	SD	0.001		4.67E+04
0.010	R1	0.070	1	7.83E+05
	R2	0.066	7	5.82E+05
	R3	0.070	1	7.60E+05
	Mean	0.069	3	7.08E+05	15
	SD	0.002		1.10E+05
0.032	R1	0.071	0	7.99E+05
	R2	0.071	0	8.40E+05
	R3	0.069	3	7.24E+05
	Mean	0.070	1	7.88E+05	5
	SD	0.001		5.88E+04
0.10	R1	0.066	7	5.90E+05
	R2	0.066	7	5.57E+05
	R3	0.066	7	5.87E+05
	Mean	0.066	7	5.78E+05
	SD	0.000		1.78E+04	31
0.32	R1	0.030	58	3.73E+04
	R2	0.016	77	1.13E+04
	R3	0.014	80	8.73E+03
	Mean	0.020	72	1.91E+04	98
	SD	0.009		1.58E+04
1.0	R1	0.005	93	2.25E+03
	R2	0.002	97	9.84E+02
	R3	0.000	100	1.04E+02
	Mean	0.002	97	1.11E+03	100
	SD	0.003		1.08E+03

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

SD = Standard Deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and gave the following results:
As copper:
Growth Rate: EC50 0.083 mg/L; NOEC 0.038 mg/L
As copper diformate:
Growth Rate: EC50 0.20 mg/L; NOEC 0.092 mg/L