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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

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 yet, at the beginning of the study, 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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

In order to determine acute toxicity, the fish are exposed to the test item: «CuGUN» during a period of 96 hours. Mortality is recorded at 24, 48, 72 and 96 hours.
A testing strategy (OECD MonographNo. 126) was developed to reduce the use of fish for assessing hazardous properties for the aquatic environment.This strategy is included in the ECHA technical guidance R7b (November 2012) for the tests within the REACH Regulation.
A limit test is performed (in exposing 8 fish) at the lowest of the two EC50 concentrations obtained in the algal growth inhibition test and in Daphnia magna acute immobilization test (the Upper Threshold Concentration (UTC)).

GLP compliance:

yes (incl. QA statement)

Remarks:

Certifcate is included in the report (date of inspection: from 22 to 24 January 2013)

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 the test solution all 24 hours and at the renewal of the test solution.

Vehicle:

no

Details on test solutions:

The stock solution at 1000 mg/L of CuGUN has been prepared in deionised water acidified with nitric acid to reach pH < 4. Indeed, according to information provided by the sponsor, CuGUN is hydrolyzed in N-guanylurea and copper, at pH 7.
This stock solution has been diluted 1/100° to obtain a diluted solution at 10 mg/L, in dilution water.
60 mL of this solution (10 mg/L) were introduced into glass vessels containing dilution water.
The characteristics of stock solutions (1000 mg/L) and solutions at 10 mg/L were the following:

Stock solutions 1000 mg/L:
pH: 6.7
Dissolved oxygen: between 8.2 and 8.5 mg O2/L
Conductivity: betweeen 546 and 552 µS/cm

Stock solutions 10 mg/L:
pH: between 7.6 and 7.9
Dissolved oxygen: between 8.5 and 8.6 mg O2/L

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish

- Source: EXOMARC (33310 Lormont, FRANCE)
- Age at study initiation (mean and range, SD): NA
- Mean size: 2.9 cm ± 0.1 cm
- Mean wet weight: 0.45 g ± 0.04 g
- Holding:
- a single stock held in test facility since 21 January 2013
- Water: Dilution water NF EN ISO 6341 (continuous aeration and filtering device)
- Feeding: TetraMin Tetra
- Feeding frequency: every days worked
- Light: 14-hour light / 10-hour dark cycle

ACCLIMATION
- Acclimation period: > 15 days
- Acclimation conditions : same as test
- Mortality observed: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

/

Hardness:

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

Test temperature:

Between 22 and 24°C

pH:

pH at T 0
Controls: 7.9
Test solutions at 0.15 mg/L: 7.9

Dissolved oxygen:

Dissolved oxygen at T 0
Controls: between 8.3 and 8.5 mg O2/L
Test solutions at 0.15 mg/L: between 8.3 and 8.5 mg O2/L

Salinity:

Not available

Nominal and measured concentrations:

Nominal concentrations CuGUN: 0.15 mg/L

Copper and N-guanylurea measured concentrations in control and in test concentration (see table 1).

Amount of element measured compared to the theoretical amount of element (see table 2).

 "CuGUN equivalent” concentrations (see table 3).

Details on test conditions:

Vessel: 5 litres glass vessels filled with 4 litres
Test: 2 blank controls + 2 test vessels
Number of animals: 8 for test concentration and 8 for the control
Test concentration: 0.15 mg/L (initial nominal concentration)
Biomass: 0.45 g/L
Feeding: none
Light: 14-hour light / 10-hour dark cycle
Aeration: none
Renewal: after 48 hours (test concentration and control)
Duration: 96 hours
Times of observation: 3 - 6 - 24 – 48 – 72 - 96 hours

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 0.1 mg/L

Conc. based on:

other: copper and N-guanylurea

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.1 mg/L

Conc. based on:

other: copper and N-guanylurea

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.1 mg/L

Conc. based on:

other: copper and N-guanylurea

Details on results:

Calculations of “CuGUN equivalent” concentrations on the basis of copper and N-guanylurea led to slightly different concentrations values.

Therefore, two concentrations have been calculated with “CuGUN equivalent” concentrations:
- Based on total copper measurement: 0.10 mg/L
- Based on N-guanylurea measurement: 0.11 mg/L

In conditions of the test, no lethal or sublethal effect on Danio rerio has been observed, for the test item CuGUN at 0.10 mg/L, after 96 hours.

Results with reference substance (positive control):

Test result for the selected batch (test carried out from 07 February 2013 to 08 February 2013):
LC50 24h = 224.0 mg/L (205.2 – 244.2)
The LC50 24 hours was calculated using a logistic regression analysis with a bootstrap estimation of the 95% confidence limits.
This value is included in the warning limits of the laboratory (mean LC50 +/- 2 SD) on this species corresponding to two standard deviations from the historic geometric mean of the median lethal concentration (131.9 – 296.0 mg/L). This result confirms the appropriate sensitivity of the fish batch.

Reported statistics and error estimates:

/

Validity criteria fulfilled:

yes

Remarks:

The mortality observed in control vessels did not exceed 10% at end of the test. The dissolved oxygen concentration was at least 60% of air saturation value in control vessels and in test vessels throughout the test period.

Conclusions:

The value of LC50 Danio rerio will be greater than the EC50 value obtained for the most sensitive species (i.e. Daphnia magna: EC50 48h: 0.084 mg/L “equivalent CuGUN” based on total copper measurement and EC50 48h: 0.132 mg/L “equivalent CuGUN” based on N-guanylurea measurement).

Therefore:
- There is no need to carry out a full test on Danio rerio
- The LC50 Danio rerio is greater than the EC50 value obtained for the most sensitive species (Upper Threshold Concentration, obtained on Daphnia magna).

Executive summary:

In order to determine acute toxicity, the fish are exposed to the test item: «CuGUN» during a period of 96 hours. Mortality is recorded at 24, 48, 72 and 96 hours.

A testing strategy (OECD MonographNo. 126) was developed to reduce the use of fish for assessing hazardous properties for the aquatic environment.This strategy isincluded in the ECHA technical guidance R7b (November 2012) for the tests within the REACH Regulation. A limit test is performed (in exposing 8 fish) at the lowest of the two EC₅₀ concentrations obtained in the algal growth inhibition test and in Daphnia magna acute immobilization test (the Upper Threshold Concentration (UTC)).

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).

The value of LC₅₀ Danio rerio will be greater than the EC₅₀ value obtained for the most sensitive species (i.e. Daphnia magna: EC₅₀ 48h: 0.084 mg/L “equivalent CuGUN” based on total copper measurement and EC₅₀ 48h: 0.132 mg/L “equivalent CuGUN” based on N-guanylurea measurement).

Description of key information

In order to determine acute toxicity, the fish are exposed to the test item: «CuGUN» during a period of 96 hours. Mortality is recorded at 24, 48, 72 and 96 hours. 
A testing strategy (OECD MonographNo. 126) was developed to reduce the use of fish for assessing hazardous properties for the aquatic environment.This strategy isincluded in the ECHA technical guidance R7b (November 2012) for the tests within the REACH Regulation. A limit test is performed (in exposing 8 fish) at the lowest of the two EC50 concentrations obtained in the algal growth inhibition test and in Daphnia magna acute immobilization test (the Upper Threshold Concentration (UTC)). 
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). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.1 mg/L

Additional information

Calculations of “CuGUN equivalent” concentrations on the basis of copper and N-guanylurea, products of degradation of the test item, led to slightly different concentrations values.

Therefore, two concentrations have been calculated with “CuGUN equivalent” concentrations: - Based on total copper measurement: 0.10 mg/L - Based on N-guanylurea measurement: 0.11 mg/L.

In conditions of the test, no lethal or sublethal effect on Danio rerio has been observed, for the test item CuGUN at 0.10 mg/L, after 96 hours.

The value of LC50 Danio rerio will be greater than the EC50 value obtained for the most sensitive species (i.e. Daphnia magna: EC50 48h: 0.084 mg/L “equivalent CuGUN” based on total copper measurement and EC50 48h: 0.132 mg/L “equivalent CuGUN” based on N-guanylurea measurement).