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

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:

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 202 (Daphnia sp. Acute Immobilisation 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 initial concentration and maintenance of the exposure concentrations during the test have been checked by analyzing hydrolysis products (copper and N-guanylurea).
Analyses have been performed on each test solutions at the beginning and the end of the test.

At T48h, for each test concentration and for the control, analysis has been conducted, on solutions containining daphnids ( by pooling the eight vessels for each test concentration)

Vehicle:

no

Details on test solutions:

A stock solution of CuGUN at 10 mg/L was prepared in deionised water acidified (pH<4) with nitric acid. This stock solution has been diluted ten times to obtain a solution at 1 mg/L in dilution water. Appropriate quantities of this solution (1 mg/L) were introduced into the glass vessel containing dilution water, to reach the required concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus 1820
- Source: INERIS
- Age at study initiation (mean and range, SD): less than 24 hours

- Feeding during test: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

NONE

Hardness:

Dilution water (NF EN ISO 6341): 240 mg/L expressed as CaCO3

Test temperature:

20°C+/- 1°C

pH:

pH at T 0h

Control : 7.5

Stock solution (10 mg/L): 4.0

Diluted solution (1 mg/L): 7.1

Dissolved oxygen:

Dissolved oxygen at T 0h

Control : 8.7 mg/L

Stock solution (10 mg/L): 7.7 mg O2/L;

Diluted solution (1 mg/L): 8.4 mg O2/L

Nominal and measured concentrations:

Measurements à T 0h : see table 4
Measurements à T 48h : see table 5

Details on test conditions:

TEST SYSTEM
- Test vessel: disposable glass tubes of 20 mL, filled with 10 mL of test solution
- Type : open
- Aeration: None

- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 8
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Composition of the dilution water (NF EN ISO 6341)
Chemicals Amount in 1 litre of dilution water
CaCl2, 2H2O 294.0 mg
MgSO4, 7H2O 123.3 mg
NaHCO3 64.75 mg
KCl 5.75 mg
All the chemicals were of recognised analytical grade purity.

- Alkalinity: Not measured
- Ca/Mg ratio: 4
- Conductivity: 647 µS/cm
- Dissolved oxygen concentration: 8.7 mg/L
- Culture medium different from test medium: Yes M4 Elendt


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: No
- Light intensity: Complete darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility at 24 and 48 hours

TEST CONCENTRATIONS
Range finding study
- Test concentrations: mg/L 1.00 - 0.33 - 0.11 - 0.037 - 0.004 (CuGUN initial nominal concentrations)


Definitive study
-Spacing factor for test concentration: 2
Test concentrations: mg/L 0.300 - 0.150 - 0.075 - 0.038 - 0.019 - 0.009 (CuGUN initial nominal concentrations)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.084 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

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

Basis for effect:

mobility

Remarks on result:

other: 0.072 - 0.097

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.132 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

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

Basis for effect:

mobility

Remarks on result:

other: 0.114 - 0.150

Details on results:

- Behavioural abnormalities: None

- Other biological observations: None

- Mortality of control: None

- Observation at 24h : Table 6

- Observation at 48h : 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 copper concentration *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 10

Calculated values of "CuGUN equivalent" , based on hydrolysis product measurement were outside the range of 80 to 120% . Therefore, EC50 have been calculated using "CuGUN equivalent" concentration geometric mean (see table 11)

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Mobility 24h
- EC50: 1.23 mg/L (1.16 – 1.32 mg/L)
- Other: 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.

Table 6 : Observation at T 24 Hours

Test concentrations (CuGUN initial nominal concentrations) mg/L	Number of daphnids immobilised per test vessel									% of immobilisation
	A	B	C	D	E	F	G	H	Total (/40)
Control	0	0	0	0	0	0	0	0	0	0
0.009	0	0	0	0	0	0	0	0	0	0
0.019	0	0	0	0	0	0	0	0	0	0
0.038	0	0	0	0	0	0	0	0	0	0
0.075	0	0	0	0	0	0	0	0	0	0
0.150	0	0	0	0	0	0	0	0	0	0
0.300	5	5	5	5	5	5	5	5	40	100

Table 7: Observation at T 48 Hours

Test concentrations (CuGUN initial nominal concentrations) mg/L	Number of daphnids immobilised per test vessel									% of immobilisation
	A	B	C	D	E	F	G	H	Total (/40)
Control	0	0	0	0	0	0	0	0	0	0
0.009	0	0	0	0	0	0	0	0	0	0
0.019	0	0	0	0	0	0	0	0	0	0
0.038	0	0	0	0	0	0	0	0	0	0
0.075	1	2	0	0	1	0	1	1	6	15
0.150	4	4	5	4	3	5	3	1	29	72.5
0.300	5	5	5	5	5	5	5	5	40	100

Table 8: pH at T 48h

Control	Test item 0.038 mg/L	Test item 0.300 mg/L
7.7	7.7	7.7

Table 9: Dissolved oxygen at T 48h

Control	Test item 0.038 mg/L	Test item 0.300 mg/L
8.3	8.4	8.1

Table 10 : “CuGUN equivalent” concentrations based on total copper measured concentration and onN-guanylurea measured concentration.

CuGUN initial nominal concentration (mg/L)	T 0h		T48h
	CuGUN equivalent concentration (mg/L) based on
	Total copper	N-guanyl urea	Total copper	N-guanyl urea
0.009	0.005	<0.010	0.008	0.011
0.019	0.016	0.018	/	0.022
0.038	0.029	0.049	0.030	0.044
0.075	0.058	0.093	0.051	0.089
0.150	0.110	0.173	0.107	0.152
0.300	0.227	0.457	0.210	0.329

Table 11 : Geometric mean of “CuGUN equivalent” based on total copper and N-guanylurea measured concentrations

CuGUN initial nominal concentration (mg/L)	Geometric mean of“CuGUN equivalent”
	based on Total copper	based onN-guanyl urea
0.009	0.006	/
0.019	/	0.020
0.038	0.029	0.046
0.075	0.054	0.091
0.150	0.108	0.162
0.300	0.218	0.388

Validity criteria fulfilled:

yes

Remarks:

In the control vessels, the immobilisation 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 test vessels.

Conclusions:

The test is valid, an EC50 (48h) of 0.084 mg/L was determined.
According to the Regulation EC 1272/2008, Copper Guanylurea nitrate as to be classified as Aquatic Acute 1.

Executive summary:

Young daphnids, aged less than 24 hours at the start of the test, have been exposed to the test item CuGUN at a range of concentrations between 0.009 mg/L and 0.300 mg/L for a period of 48 hours.

Immobilisation was recorded at 24 hours and 48 hours.

CuGUN was hydrolyzed, in dilution water, in copper and N-guanyl urea. Then, the exposure concentrations during the test have been checked by analyzing hydrolysis products (copper et N-guanyl urea) at T 0h and T 48h.

EC₅₀ of immobilisation have been calculated using "CuGUN equivalent" concentrations

	EC5048h  “CuGUN equivalent”concentrations (Confidence interval at 95%)
based on total copper measured concentrations.	0.084 mg/L (0.072 – 0.097)
based onN-guanyl urea measured concentrations.	0.132 mg/L (0.114 – 0.150)

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

Description of key information

Young daphnids, aged less than 24 hours at the start of the test, have been exposed to the test item CuGUN at a range of concentrations between 0.009 mg/L and 0.300 mg/L for a period of 48 hours. 
Immobilisation was recorded at 24 hours and 48 hours. 
CuGUN was hydrolyzed, in dilution water, in copper and N-guanyl urea. Then, the exposure concentrations during the test have been checked by analyzing hydrolysis products (copper et N-guanyl urea) at T 0h and T 48h.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.084 mg/L

Additional information

CuGUN was hydrolyzed, in dilution water, in copper andN-guanylurea. Therefore, EC₅₀48h have been calculated based on measured concentrations of copper andN-guanylurea.

 

	EC5048h “CuGUN equivalent” concentration (Confidence interval at 95%)
Based on total copper measured concentrations.	0.084 mg/L (0.072 – 0.097)
Based onN-guanyl urea measured concentrations.	0.132 mg/L (0.114 – 0.150)