Bis(glycinato)copper

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

The experiment was conducted before April 1984.

Deviations:

yes

Remarks:

Deviations: Oxygen was not measured. 12 h light, 12 h dark instead of 16 h/8 h.

Principles of method if other than guideline:

Although conducted before April 1984, the experiment reported in this study fulfills the main criteria of the OECD guideline 202.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

not described

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna
- Age at study initiation: 0 to 24 h
- Food type: no feeding during study

Test type:

other: static test conditions are assumed according to the experimental description

Water media type:

other: artificial medium containing 1 mM CaCl2, 2 mM NaHCO3 and 5 mM NaCl in distilled water at a ionic strength of 0.01.

Total exposure duration:

48 h

Hardness:

2 mM NaHCO3

Test temperature:

20 ± 1 °C

pH:

8.37 to 8.43

Nominal and measured concentrations:

not applicable

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: glass, 250 mL
- Volume of solution: 200 mL
- Aeration: yes, overnight
- No. of organisms per vessel: 15 to 30
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water
- Ca/mg ratio: medium did not contain Mg
OTHER TEST CONDITIONS
- Adjustment of pH: yes, to keep as closely to 8.4 as possible
- Photoperiod: 12/12
- Light intensity: not reported
EFFECT PARAMETERS MEASURED: EC50, immobilization
VEHICLE CONTROL PERFORMED: not applicable
RANGE-FINDING STUDY: no

Reference substance (positive control):

yes

Remarks:

comparison to free copper ions

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.18 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Cu2+ ions complexed with glycine

Basis for effect:

mobility

Details on results:

No effects other than immobilization were reported.

Results with reference substance (positive control):

The reported EC50 of the copper-glycine complex (10.3 µM) was substantially higher compared to the EC50 of free copper ions (0.501 µM).

Reported statistics and error estimates:

The percentage of mobile was plotted against log total copper concentration, and the EC50 estimated from the intercept at 50 % for the two observations just above and below 50 % mobility. Probit analysis could not be used because survival occasionally decreased from 100 to <50 or from >50 to 0 % over one concentration interval. The mean and 95 % confidence limits for the EC50 were calculated from a minimum of 4 (usually 6 or more) separate EC50s from separate experiments.

The toxicity of several copper-amino acid complexes to aquatic organisms was compared to that of free copper ions.

Glycine, amongst other complexing agents, was added to solutions containing free copper ions and the rate of complex formation was monitored. It was demonstrated that most of the copper present was bound to glycine and thus, total copper EC₅₀ values were approximately equal to the concentration of the copper/glycine complexes (Table 1). Addition of 200 µM glycine lead to more than 97 % reduction of the initial free copper concentration.

It was proven that complexation with glycine reduced the EC₅₀ of free copper ions more than 20-fold and thus the toxicity to aquatic invertebrates (Daphnia magna).

Table 1: EC₅₀ increase after addition of glycine and free copper concentrations from strictly chemical measurements and their 95 % confidence limits.

	EC50 48 h [µM]	EC50 48 h [mg/L]	Free copper (determined by analysis) [nM]
Inorganic medium	0.501	0.11	6.99 (4.59-10.64)
Inorganic medium plus 200 µM glycine	10.3	2.18	0.189 (0.130- 0.276)

Validity criteria fulfilled:

not applicable

Conclusions:

In this study similar to guideline OECD 202, the EC50 of the test item towards Daphnia magna was determined to be 2.18 mg/L. Basis for effect was immobilization.

Executive summary:

The publication of Borgmann and Ralph (1983) describes the reduction of copper toxicity to Daphnia magna, as determined by EC₅₀, after complexation with various agents like glycine. Though the study was conducted before 1984, the experimental principles are considered similar to OECD guideline 202.

Daphnia magna were exposed to free copper ions at 20 °C without food and under a 12 h light / 12 h dark photoperiod for 48 h. A logarithmic concentration series was used following the series 10, 13, 18, 24, 32, 42, 56, 75 and 100. The EC₅₀ was determined in inorganic media with and without addition of 200 µM glycine. The pH was adjusted to 8.4. Tests were conducted using 0-24 h old animals and the number still mobile after 48 h was recorded. The complexation rate was determined by analysis of residual free copper after addition of glycine by atomic absorption spectrophotometry. It was demonstrated that most of the copper present was bound to glycine. Addition of 200 µM glycine lead to more than 97 % reduction of the initial free copper concentration.

Furthermore, it was proven that complexation with glycine reduced the EC₅₀ more than 20-fold compared to free copper ions. The authors describe that the complex of copper and glycine has an EC₅₀ of 10.3 µM.

The EC₅₀ of the copper-glycine complex towards Daphnia magna was thus determined to be 2.18 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 1984 to January 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: according to the method described for the static test by the Committee on Methods for Toxicity Test with Aquatic Organisms.

Version / remarks:

1975

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

distilled water

Test organisms (species):

Daphnia magna

Details on test organisms:

- Daphnia magna were initially collected from the natural pond situated in Gheru Campus and a stock culture was made in well water.
- Feeding during test: no
- Food type during culture: fish food chow and algae

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

240 ppm as CaCO3

Test temperature:

13 ± 2 °C

pH:

7.6

Dissolved oxygen:

>= 5 mg/L

Nominal and measured concentrations:

no measurement

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Fill volume: 200 mL
- Volume of solution: 100 mL
- Aeration: yes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark according to Committee on Methods for Toxicity Test with Aquatic Organisms (1975), but not clearly reported in the publication
EFFECT PARAMETERS MEASURED: The criteria for determining the death were complete immobilization, stop of hearts beats and no response to gentle prodding with blunt glass rod. Daphnids were observed at 30 min intervals during the first 4 to 6 hr and hourly up to 6 to 8 hr. Additional observations were made at 4 to 6 hr intervals for remainder period of each test. Observations were continued until all animals were dead or until of the end of 48 hr.

Reference substance (positive control):

yes

Remarks:

free copper ions added as copper sulfate pentahydrate

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.129 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Cu2+ complexed with glycine

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

3.346 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Cu2+ complexed with glycine

Basis for effect:

mortality

Details on results:

- the following effects were observed: immobilisation, stop of heartbeat and response to gentle prodding with a blunt glass rod.
- addition of 10 mg/L glycine led to a more than 20-fold increase of the LC50.

Results with reference substance (positive control):

- LC50 of Cu2+ added as copper sulfate pentahydrate (48 h): 0.093 mg/L

Reported statistics and error estimates:

The results of three tests were combined and the 24 and 48 hr LC50 values and tests of significant difference were calculated according to Harris (1959). The median survival time and 95% confidence limits were calculated by the methods of Litchfield (1949).

The toxicity of several copper-amino acid complexes to Daphnia magna was compared to that of free copper ions. 10 mg/L glycine, amongst other amino acids, was solved in solutions containing free copper ions added as copper sulfate pentahydrate and the difference in LC₅₀ determined.

It was proven that the addition of glycine reduced the LC₅₀ of free copper ions more than 20-fold and thus decreases the toxicity of copper to Daphnia magna.

The reduction in copper toxicity is supposed to be due to the formation of a copper-amino acid complex less toxic than copper alone in test solution.

Addition of glycine caused the highest decrease in copper toxicity to daphnids in these experiments. Generally, all tested amino acids caused a marked decrease in copper toxicity. In comparison, a copper concentration of 0.56 mg/L and above caused 100 % mortality in 48 h. At 0.32 and 0.1 mg/L of Cu alone, 90% and 20% mortality were noticed.

Table 1: Comparison of LC₅₀ of free and glycine complexed copper. 95 % confidence intervals are displayed in brackets.

	LC50 48 h [mg/L]	Fold reduction of LC50
Cu^2+	0.093 (0.081-0.112)
Cu^2+ complexed with 10 mg/L glycine	2.129 (1.642-2.805)	> 22

Validity criteria fulfilled:

not applicable

Conclusions:

In this study, the LC50 of the test item towards Daphnia magna was determined to be 2.129 mg/L.

Executive summary:

In the publication of Khangarot et al. (1987), the reduction of copper toxicity to Daphnia magna, as determined by LC₅₀, was assessed after complexation of free copper ions with glycine and other amino acids. The study was conducted according to the method described for the static test by the Committee on Methods for Toxicity Test with Aquatic Organisms (1975) and is thus considered reliable.

Daphnia magna were collected from a natural pond and a stock culture was established. Daphnids were exposed to free copper ions in form of copper sulfate pentahydrate at 13 ± 2 °C without food for 48 h. The LC₅₀ was determined in triplicates with and without addition of 10 mg/L glycine. The pH was 7.6. Dissolved oxygen was at least 5 mg/L. The criteria for determining the death were complete immobilization, stop of heartbeats and no response to gentle prodding with blunt glass rod.

All tested amino acids caused a marked decrease in copper toxicity. The authors state that addition of glycine to the copper solution caused the highest decrease in copper toxicity to daphnids. It was shown that complexation with glycine reduced the LC₅₀ more than 20-fold compared to free copper ions.

In conclusion, the 48 h LC₅₀ of a copper solution supplemented with 10 mg/L glycine towards Daphnia magna was determined to be 2.129 mg/L.

Description of key information

- Borgmann and Ralph (1983): comparable to guideline, RL1: The EC₅₀ of the copper-glycine complex towards Daphnia magna was determined to be 2.18 mg/L.

- Khangarot et al. (1987): comparable to guideline, RL2: the 48 h LC₅₀ of a copper solution supplemented with 10 mg/L glycine towards Daphnia magna was determined to be 2.129 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.18 mg/L

Additional information

The publication of Borgmann and Ralph (1983) describes the reduction of copper toxicity to Daphnia magna, as determined by EC₅₀, after complexation with various agents like glycine. Though the study was conducted before 1984, the experimental principles are considered similar to OECD guideline 202. Daphnia magna were exposed to free copper ions at 20 °C without food for 48 h. A logarithmic concentration series was used following the series 10, 13, 18, 24, 32, 42, 56, 75 and 100. The EC₅₀ was determined in inorganic media with and without addition of 200 µM glycine. The pH was adjusted to 8.4. Tests were conducted using 0-24 h old animals and the number still mobile after 48 h was recorded. The complexation rate was determined by analysis of residual free copper after addition of glycine by atomic absorption spectrophotometry. It was demonstrated that most of the copper present was bound to glycine. Addition of 200 µM glycine lead to more than 97 % reduction of the initial free copper concentration. Furthermore, it was proven that complexation with glycine reduced the EC₅₀ more than 20-fold compared to free copper ions. The authors describe that the complex of copper and glycine has an EC₅₀ of 10.3 µM. The EC₅₀ of the copper-glycine complex towards Daphnia magna was thus determined to be 2.18 mg/L.

In the publication of Khangarot et al. (1987), the reduction of copper toxicity to Daphnia magna, as determined by LC₅₀, was assessed after complexation of free copper ions with glycine and other amino acids. The study was conducted according to the method described for the static test by the Committee on Methods for Toxicity Test with Aquatic Organisms (1975) and is thus considered reliable. Daphnia magna were collected from a natural pond and a stock culture was established. Daphnids were exposed to free copper ions in form of copper sulfate pentahydrate at 13 ± 2 °C without food for 48 h. The LC₅₀ was determined in triplicates with and without addition of 10 mg/L glycine. The pH was 7.6. Dissolved oxygen was at least 5 mg/L. The criteria for determining the death were complete immobilization, stop of heartbeats and no response to gentle prodding with blunt glass rod. All tested amino acids caused a marked decrease in copper toxicity. The authors state that addition of glycine to the copper solution caused the highest decrease in copper toxicity to daphnids. It was shown that complexation with glycine reduced the LC₅₀ more than 20-fold compared to free copper ions. In conclusion, the 48 h LC₅₀ of a copper solution supplemented with 10 mg/L glycine towards Daphnia magna was determined to be 2.129 mg/L.

The test item is considered to be toxic to aquatic invertebrates according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).