Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Analysis of the test item was performed by the chemical analysis unit of INERIS (CARA/RESA) that is not included in the GLP areas of expertise of the Test Facility. This analytical phase has been inspected by the Quality Assurance Personnel of the Test Facility for both the technical phase and the raw data generated. Consequently, owing to the approach set up for the control of these data, we estimate that the analytical monitoring of the test item does not affect compliance with Good Laboratory Practices in this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

CuGUN is rapidly hydrolyzed in test medium, in copper and N-guanylurea.
Then, the exposure concentrations during the test have been checked by following hydrolysis products (copper and N-guanylurea).
Analyses have been performed on each test solutions at the beginning and the end of the test.
At T72h, for each test solution and for the control, analyses have been performed, after algal cells number determination, on pool of the three replicates solutions.

Vehicle:

no

Details on test solutions:

The stock solution at 10 mg/L was prepared in deionised water acidified with nitric acid to reach pH ≤ 4. Appropriate quantities of this stock solution have been introduced into the glass vessels containing growth medium to reach required test concentrations.

Test organisms (species):

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

Details on test organisms:

Strain: CCAP 278/4
Liquid culture of Pseudokirchneriella subcapitata was obtained from the Culture Center for Algae and Protozoa (CCAP) Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria, U.K.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

23 ± 2°C

pH:

Growth medium pH : 8.2 ± 0.2

Nominal and measured concentrations:

Measurement at T 0h: see table 4
Measurement at T 72h: see table 5

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml conical flasks with air permeable stoppers filled with 100 ml of test concentration
- Aeration: none.
- Renewal rate of test solution : none
- Initial cells density: 10 E04 cells/ mL taken from an exponentially growing preculture.
- Control end cells density: 103.37 E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Blank control: 1 replicate without algae by concentration

- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition

Stock solutions
Stock solution 1:
- NH4Cl 1.5 g/L
- MgCl2 - 6H2O 1.2 g/L
- CaCl2 - 2H2O 1.8 g/L
- MgSO4 - 7H2O 1.5 g/L
- KH2PO4 0.16 g/L

Stock solution 2:
- FeCl3 - 6H2O 64 mg/L
- Na2EDTA - 2H2O 100 mg/L

Stock solution 3:
- H3BO3 185 mg/L
- MnCl2 - 4H2O 415 mg/L
- ZnCl2 3 mg/L
- CoCl2 - 6H2O 1.5 mg/L
- CuCl2 - 2H2O 0.01 mg/L
- Na2MoO4 - 2H2O 7 mg/L
Stock solution n°4:
- NaHCO3 50 g/L
Stock solutions 1 and 3 were sterilized by autoclaving (120°C, 20 min). Stock solutions 2 and 4 were sterilized by membrane filtration (0.22 µm).
The growth medium was prepared as follows:
Stock solution 1 10 mL
Stock solution 2 1 mL
Stock solution 3 1 mL
Stock solution 4 1 mL
Sterile deionised water qsp 1000 mL
After equilibration with air, the pH of growth medium was 8.2 ± 0.1.

if non-standard medium was used:


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality : white light, intensity within the range 6200 – 8300 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, every 24 hours


TEST CONCENTRATIONS
Range finding study
- Test concentrations: 10 - 3.125 - 0.976 - 0.305 - 0.095 - 0.03 (CuGUN initial nominal concentration)
- Results used to determine the conditions for the definitive study:3 % inhibition at 0.03 mg/L, 81.4% inhibition at 0.976 mg/L (CuGUN initial nominal concentration )

Defintive test
- Spacing factor for test concentrations: 1.8
- Test concentrations: 1.00 - 0.56 - 0.31 - 0.17 - 0.10 - 0.05 (CuGUN initial nominal concentration)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.174 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured total copper concentration

Basis for effect:

growth rate

Remarks on result:

other: 0.153 - 0.210

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.234 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured N-guanylurea concentration

Basis for effect:

growth rate

Remarks on result:

other: 0.197 - 0.286

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured total copper concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured N-guanylurea concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.062 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured total copper concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.069 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: "CuGUN equivalent" concentration based on measured N-guanylurea concentration

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
Cells density measurements are presented in table 6
Growth rates calculations are presented in table 7


As CuGUN is hydrolizing in copper and N-guanyl urea, "CuGUN equivalent" concentrations have been calculated, based on :
-Total copper measured concentration (CUGUN equivalent = Total copperconcentration *CuGUN molar mass /Copper molar mass)
-N-guanylurea concentration (CUGUN equivalent = N-guanylurea concentration *CuGUN molar mass/ 2*N-guanylurea molar mass)

with :
CuGUN molar mass = 391.75 g/mol
Copper molar mass = 63.5 g/mol
N-guanyl molar mass = 102 g/mol
See Table 8
The analyses of the two hydrolysis products led to determine different values for “CuGUN equivalent” for each concentration. “CuGUN equivalent” concentration and nominal concentration have been compared : measured concentrations of total copper are reflecting 61% to 107% of nominal concentrations of CuGUN, and measured concentrations of N-guanylurea are corresponding to 100% to 140% of nominal concentrations of CuGUN.
Rationale for such a difference of rate between total copper and N-guanylurea was not identified as uncertainties on analysis of the two hydrolysis products couldn’t explain it. As no certificate of analysis was available for the batch of the tested item, differences between general characteristics provided by the sponsor and the tested item (e.g. purity) could be an explanation for these measured values.
Considering these results, the use of nominal values for EC10 and EC50 72h calculations was not relevant (calculated values, based on hydrolysis products measurements were outside the range of 80 to 120%).
Therefore, EC10 and EC50 have been calculated using "CuGUN equivalent" concentration geometric mean (see table 9)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.07 mg/L (0.96 - 1.22 mg/L)

Reported statistics and error estimates:

EC50 72h have been calculated using log-logistic regression with bootstrap estimation of the 95% confidence limits
NOECs have been determined with the following statistical strategy : ANOVA and Bonferroni test (Toxstat 3.5 ,Western Inc. and D. Gulley, University of Wyoming)

Table 6 : Cells density measurements

Test Concentrations (in CuGUN initial nominal concentration)	Cell Density x 10^4cell.ml^-1(particle counter)								pH
	T0	Mean	T24h	Mean	T48h	Mean	T72h	Mean	T0	T72h
Controls	1.00 1.00 1.00 1.00 1.00 1.00	1.00	3.93 3.91 4.04 6.08 4.12 3.38	4.24	17.16 21.57 15.72 16.26 17.36 19.88	17.99	110.54 129.90 83.19 116.87 82.98 96.76	103.37	8.1	8.0 8.2 7.8 8.0 7.9 7.9
0.05	1.00 1.00 1.00	1.00	2.83 3.53 2.99	3.12	20.36 18.90 17.10	18.79	131.33 131.04 101.21	121.19	8.1	8.5
0.10	1.00 1.00 1.00	1.00	2.94 3.06 3.02	3.01	17.82 14.66 14.67	15.72	111.79 93.67 77.93	94.46	8.1	8.0
0.17	1.00 1.00 1.00	1.00	2.66 3.19 2.61	2.82	4.37 4.52 5.13	4.67	8.54 11.22 12.06	10.61	8.1	7.8
0.31	1.00 1.00 1.00	1.00	2.61 2.39 2.29	2.43	2.89 3.02 3.35	3.09	3.59 3.49 3.91	3.66	8.1	7.7
0.56	1.00 1.00 1.00	1.00	2.39 2.18 2.07	2.21	2.51 2.32 2.77	2.53	3.28 3.03 2.57	2.96	8.1	7.7
1.00	1.00 1.00 1.00	1.00	1.99 1.96 2.42	2.12	2.68 2.26 2.71	2.55	2.45 2.83 2.43	2.57	8.0	7.7

Table 7 : Growth rates calculations

Test concentrations (CuGUN Initial nominal concentration)	Average specific growth rate (days^–1)									Inhibition of growth rate
	0-24h	Mean	24-48h	Mean	48-72h	Mean	0-72h	Mean	sd
Controls	1.369 1.363 1.396 1.805 1.416 1.218	1.428	1.474 1.709 1.359 0.983 1.439 1.772	1.456	1.863 1.795 1.666 1.972 1.564 1.583	1.741	1.568 1.622 1.474 1.587 1.473 1.524	1.541	0.062	/
0.05	/	/	/	/	/	/	1.626 1.625 1.539	1.597	0.050	-3.6%
0.10	/	/	/	/	/	/	1.572 1.513 1.452	1.512	0.060	1.9%
0.17	/	/	/	/	/	/	0.715 0.806 0.830	0.784	0.061	49.2%
0.31	/	/	/	/	/	/	0.426 0.417 0.454	0.432	0.019	71.9%
0.56	/	/	/	/	/	/	0.396 0.369 0.314	0.360	0.042	76.7%
1.00	/	/	/	/	/	/	0.298 0.346 0.296	0.313	0.028	79.7%

Table 8 : "CuGUN equivalent" concentrations based on total copper and N-guanylurea measured concentrations.

CuGUN initial nominal concentration (mg/L)	T 0h		T72h
	CuGUN equivalent concentration (mg/L) based on
	Total copper	N-guanyl urea	Total copper	N-guanyl urea
50	0.038	0.048	0.054	0.069
100	0.078	0.108	0.107	0.114
170	0.130	0.172	0.158	0.188
310	0.221	0.340	0.261	0.347
560	0.401	0.653	0.394	0.625
1000	0.613	1.095	0.673	1.062

Table 9 : Geometric mean of "CuGUN equivalent" concentrations based on total copper and N-guanylurea measured concentrations.

CuGUN initial nominal concentration (µg/L)	Geometric mean of“CuGUN equivalent” mg/L
	based on Total copper	based onN-guanyl urea
50	0.045	0.057
100	0.091	0.111
170	0.143	0.180
310	0.240	0.343
560	0.397	0.639
1000	0.643	1.078

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria
•The biomass in the control cultures increased by a factor of more than 16, corresponding to a specific growth rate of 0.92 day-1(mean cell concentration at T 0h: 1.00 E04 cell/.mL ; mean cell concentration at T 72h: 103.37 E04 cell/mL).
•The mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures did not exceed 35 % (mean coefficient of variation: 11.2 %).
•The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (coefficient of variation: 3.99%).

An EC50 (48h) of 0.174 mg/L was determined.
According to the Regulation EC 1272/2008, Copper Guanylurea nitrate as to be classified as Aquatic Acute 1.

Executive summary:

Exponentially growing cells of unicellular green algaePseudokirchneriella subcapitata have been exposed to the test item CuGUN at a range of concentrations between 0.05 an 1.00 mg/L for a period of 72 hours, under continuous illumination. Algal cell concentrations have been determined every 24 hours and inhibition has been measured as a reduction in growth rate relative to control cultures grown under identical conditions.

CuGUN was hydrolyzed in copper and N-guanylurea. Then the exposure concentrations during the test have been checked by analyzing hydrolysis products (copper and N-guanylurea) at T 0h and T 72h.

ECr50 and NOECs have been determined using CuGUN equivalent concentrations

ECr50 72h of the test item CuGUN have been calculated equal to:

                  0.174 mg/L (0.153 – 0.210) “equivalent CuGUN” based on total copper determination

                  0.234 mg/L (0.197 – 0.286) “equivalent CuGUN” based on N-guanylurea determination

 

NOEC of the test item CuGUN have been determinate equal to 0.09 and 0.110 mg/L of “equivalent CuGUN” based on respectively total copper and N-guanylurea measured concentrations.

According to the Regulation EC 1272/2008, Copper Guanylurea nitrate as to be scored as Aquatic Acute 1.

Description of key information

Exponentially growing cells ofunicellular green algaePseudokirchneriella subcapitatahave been exposed to the test item CuGUN at a range of concentrations between 0.05 an 1.00 mg/L for a period of 72 hours, under continuous illumination. Algal cell concentrations have been determined every 24 hours and inhibition has been measured as a reduction in growth rate relative to control cultures grown under identical conditions.
CuGUN was hydrolyzed in copper and N-guanylurea. Then the exposure concentrations during the test have been checked by analyzing hydrolysis products (copper andN-guanylurea) at T 0h and T 72h.
ECr50 and NOECs have been determined using CuGUN equivalent concentrations

Key value for chemical safety assessment

EC50 for freshwater algae:

0.174 mg/L

EC10 or NOEC for freshwater algae:

0.09 mg/L

Additional information

ECr50 72h of the test item CuGUN have been calculated equal to:

                  0.174 mg/L(0.153 – 0.210) “equivalent CuGUN” based on total copper determination

                  0.234 mg/L(0.197 – 0.286) “equivalent CuGUN” based onN-guanylureadetermination

 

NOEC of the test item CuGUN have been determinate equal to 0.09 and 0.110 mg/L of “equivalent CuGUN” based on respectively total copper andN-guanylurea measured concentrations.