Copper glycinate sulfate

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance copper monoglycinate sulfate and the source substances copper sulfate and copper bisglycinate consist of the Cu2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance copper monoglycinate sulfate and the source substance copper bisglycinate.
It is generally accepted that the Cu2+ cation (as measure for dissolved copper species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance copper monoglycinate sulfate is a chelate-complex which consists of the divalent copper ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Copper monoglycinate sulfate and the source substance copper sulfate are ionic and consist of the Cu2+ cation and the respective anions. It is generally accepted that the copper cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, copper, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Test organisms (species):

Daphnia magna

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Borgmann and Ralph, 1983. Calculated for the target substance.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.18 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Cu2+ ions complexed with glycine

Basis for effect:

mobility

Remarks on result:

other: Borgmann and Ralph, 1983.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Kang et al., 2022. Calculated for the target substance.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.521 mg/L

95% CI:

> 0.493 - < 0.548

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Remarks on result:

other: Kang et al., 2022

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.129 mg/L

95% CI:

> 1.624 - < 2.805

Nominal / measured:

nominal

Conc. based on:

other: Cu2+ complexed with glycine

Basis for effect:

mortality

Remarks on result:

other: Khangarot et al., 1987

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Khangarot et al., 1987. Calculated for the target substance.

Validity criteria fulfilled:

not applicable

Conclusions:

The lowest expected effect value is an EC50 of copper monoglycinate sulfate dihydrate of 2.22 mg/L in Daphnia magna.

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. Although the study was conducted before 1984, the experimental principles are considered comparable 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 free copper ions towards Daphnia magna was determined to be 0.66 mg/L.The EC₅₀ of the copper-glycine complex towards Daphnia magna was determined to be 2.18 mg/L. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.79 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. 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. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.72 mg/L.

In the study by Kang et al. (2022), the acute toxicity of Cu²⁺ was assessed under conditions similar to guideline OECD 202. Daphnia magna were exposed to free copper ions in form of copper sulfate pentahydrate at 20 ± 2 °C without food for 48 h. EC10, EC20, EC50, NOEC and LOEC were determined in triplicates. The criterium for determining the death was immobilization. The 48 h EC₅₀ of copper ions (Cu²⁺) towards Daphnia magna was determined to be 0.521 mg/L. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.22 mg/L.

For precautionary reasons, the lowest expected effect value chosen is an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.22 mg/L in Daphnia magna.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

yes

Remarks:

Vehicle and test type are not clearly specified. Details on test solutions are scarce. No positive control.

GLP compliance:

not specified

Remarks:

Information on GLP is not provided in the publication.

Analytical monitoring:

no

Remarks:

Only stock solutions were analysed in triplicates for Cu concentration.

Vehicle:

not specified

Details on test solutions:

RT medium: please see section "any other details on material and methods".

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Age at study initiation: 6 – 24 h old
- Method of breeding: Daphnia magna was kept in 1 L jars containing 800 mL RT medium with pH 8, a conductivity of 270 µs/cm and hardness of 88 mg/L as CaCO3. The jars were placed at 20 ±2 °C, in a 12:12-h light/dark cycle. The animals were fed daily with the chlorophyte Scenedesmus obliquus (ca. 4 mg C/L, which was grown in WC medium.
- Source: Aquatic Ecology and Water Quality Management group of Wageningen University (The Netherlands).
- Feeding during test: no

Test type:

other: Not clearly stated. Presumably static conditions according to the description.

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 2°C

Nominal and measured concentrations:

Nominal concentrations: 0, 0.081, 0.202, 0.323, 0.404, 0.485, 0.565, 0.646, 0.808 mg Cu/L

Details on test conditions:

TEST SYSTEM
- Test vessel: jars
- Fill volume: 100 mL
- Volume of solution: 50 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 3
OTHER TEST CONDITIONS
- Photoperiod: 12/12
EFFECT PARAMETERS MEASURED: EC10, EC20, EC50, NOEC, LOEC
CONTROL PERFORMED: yes, vehicle not specified

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.521 mg/L

95% CI:

> 0.493 - < 0.548

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

0.479 mg/L

95% CI:

> 0.423 - < 0.505

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.452 mg/L

95% CI:

> 0.396 - < 0.486

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.485 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.404 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Cu2+

Basis for effect:

mobility

Reported statistics and error estimates:

The EC10, EC20 and EC50 values in the acute 48 h immobility test were analyzed by Probit analysis in the software SPSS version 25.

Chemical equilibrium modelling indicated that most copper was chelated with EDTA present in the RT medium.

100 % survival was recorded in the control series.

Validity criteria fulfilled:

no

Conclusions:

The EC50 of Cu2+ to Daphnia magna was determined to be 0.521 mg/L.

Executive summary:

In the study by Kang et al. (2022), the acute toxicity of Cu²⁺ was assessed under conditions similar to guideline OECD 202. Daphnia magna were exposed to free copper ions in form of copper sulfate pentahydrate at 20 ± 2 °C without food for 48 h. EC10, EC20, EC50, NOEC and LOEC were determined in triplicates. The criterium for determining the death was immobilization.

The 48 h EC₅₀ of copper ions (Cu²⁺) towards Daphnia magna was determined to be 0.521 mg/L.

Reference 3

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. 0.66 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Copper ion, Cu2+

Basis for effect:

mobility

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 free copper ions towards Daphnia magna was determined to be 0.66 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. Although the study was conducted before 1984, the experimental principles are considered comparable 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 free copper ions towards Daphnia magna was determined to be 0.66 mg/L.The EC₅₀ of the copper-glycine complex towards Daphnia magna was determined to be 2.18 mg/L.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key 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.:

0.093 mg/L

95% CI:

> 0.081 - < 0.112

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Copper ion, Cu2+

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.536 mg/L

95% CI:

> 0.466 - < 0.643

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Copper ion, Cu2+

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.129 mg/L

95% CI:

> 1.624 - < 2.805

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

95% CI:

> 2.855 - < 3.777

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 48 h LC50 of free copper ions was 0.093 mg Cu2+/L. 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.

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.

The 48 h LC₅₀ of free copper ions was 0.093 mg Cu²⁺/L. 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. The EC₅₀ of the target substance copper glycinate sulfate dihydrate towards Daphnia magna is therefore estimated to be 2.79 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. The LC₅₀ of the target substance copper glycinate sulfate dihydrate towards Daphnia magna is therefore estimated to be 2.72 mg/L.

- Kang et al. (2022): comparable to guideline, RL2: The 48 h EC₅₀ of copper ions (Cu²⁺) towards Daphnia magna was determined to be 0.521 mg/L.This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.22 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

ca. 2.22 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. Although the study was conducted before 1984, the experimental principles are considered comparable 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 free copper ions towards Daphnia magna was determined to be 0.66 mg/L.The EC₅₀ of the copper-glycine complex towards Daphnia magna was determined to be 2.18 mg/L. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.79 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. 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. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.72 mg/L.

In the study by Kang et al. (2022), the acute toxicity of Cu²⁺ was assessed under conditions similar to guideline OECD 202. Daphnia magna were exposed to free copper ions in form of copper sulfate pentahydrate at 20 ± 2 °C without food for 48 h. EC10, EC20, EC50, NOEC and LOEC were determined in triplicates. The criterium for determining the death was immobilization. The 48 h EC₅₀ of copper ions (Cu²⁺) towards Daphnia magna was determined to be 0.521 mg/L. This refers to an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.22 mg/L.

The results reported by Borgmann and Ralph (1983) and Khangarot et al. (1987) demonstrate that complexation with glycine substantially alleviates the toxicity of Cu²⁺ towards Daphnia magna.

For precautionary reasons, the lowest expected effect value chosen is an EC₅₀ of copper monoglycinate sulfate dihydrate of 2.22 mg/L in Daphnia magna.