Zinc 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:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A continuous flow apparatus was used for the experiments rather than a static system. For further details, please see section "any other information on materials and methods incl. tables".

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

not described

Vehicle:

no

Details on test solutions:

Concentrations of the toxicants in the mariotte bottles were set so that desired concentrations were obtained after dilution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:water flea
- Strain/clone: Daphnia magna
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: please refer to section "any other information on materials and methods incl. tables”
- Source: Arbor Scientific Supply House, Port Credit, Ontario
- Feeding during test: no

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

The dosage-mortality curves were determined for 36, 48, 60, 72, and 96 hours.

Hardness:

130 mg/L (CaCO3)

Test temperature:

20 °C (± 1)

pH:

pH = 6.95 (20 °C)

Dissolved oxygen:

number missing; 8.60 mg/L according to the corresponding thesis by Attar (1982).

Conductivity:

300 µmhos

Nominal and measured concentrations:

The concentrations ranged between 10.6 and 450.9 µg/L. Six different concentrations were tested in each experiment.

Details on test conditions:

TEST SYSTEM
- Test vessel: containers
- Material: polyethylene
- Volume of solution: 65 mL
- Aeration: not reported
- Type of flow-through: diluter
- Renewal rate of test solution (frequency/flow rate): flow through 200 mL/min
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Montreal city water
- Metals: trace amounts of cadmium (1.0 µg/L) and iron (3 µg/L), whereas zinc, nickel, and copper were not detectable.
- Alkalinity: 80 mg/L (as CaCO3)
- Conductivity: 300 µmhos
OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 12 / 12
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : LC50, 36, 48, 60, 72, and 96 hr
For a detailed description of the experimental procedure, please see section "any other information on materials and methods incl. tables".

Reference substance (positive control):

no

Duration:

36 h

Dose descriptor:

LC50

Effect conc.:

0.861 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Zn2+

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.799 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Zn2+

Basis for effect:

mortality

Duration:

60 h

Dose descriptor:

LC50

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Zn2+

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

0.126 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Zn2+

Basis for effect:

mortality

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Details on results:

- Mortality of control: zero

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The data were analysed by dosage-mortality curves (Finney 1971) and by time-mortality curves (Litchfield 1949). The dosage-mortality curves were determined for 36, 48, 60, 72, and 96 hr and were tested for similarity of slope by the F-test (Sokol and Rohlf 1969). The goodness of fit of data points to the dosage-mortality and time-mortality curves was tested with a chi-square test.

No deaths were found in controls in any experiment.

Twenty-three different zinc concentrations were tested. The calculated LC50 values of zinc for 36, 48, 60, 72, and 96 hr. are 861.06, 798.94, 420.25, 126.10, and 67.91 µg/L, respectively.

Validity criteria fulfilled:

not applicable

Conclusions:

The 48 h LC50 of zinc (Zn2+) towards Daphnia magna was 0.799 mg/L in a flow-through experiment.

Executive summary:

In the publication by Attar and Maly (1982), the toxicity of zinc towards Daphnia magna was examined in a continuous flow apparatus was used for the experiments rather than a static system. Zinc chloride (ZnCI₂) was used in this study. Metal quantities in all experimental tanks were determined daily. The concentrations ranged between 10.6 and 450.9 µg/L. Twenty-three different zinc concentrations were tested.

Observations were made every three hours. Mortality was determined by the lack of movement of the second antennules and internal organs in a five second period of observation.

No deaths were found in controls in any experiment.

The calculated LC50 values of zinc for 36, 48, 60, 72, and 96 hours were reported to be 861.06, 798.94, 420.25, 126.10, and 67.91 µg/L, respectively.

Therefore, the LC50 (48 h) of Zn²⁺ to Daphnia magna was 0.799 mg/L in this flow-through experiment.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

No data on dissolved oxygen concentration, pH and concentration of test substance after test termination.

GLP compliance:

no

Remarks:

GLP compliance was not reported in this publication.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The solutions to be tested were prepared immediately before the tests were carried out. All reagents were of analytical grade and all laboratory glassware were soaked in 10% HNO3 for at least 48h and rinsed with distilled water at least 3 times prior to use. Deionised water from a Millipore Milli-Q ultra pure (Milli- Di, France) water system was used throughout the study except for daphnid culture.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna
- Age at study initiation: < 24 h
- Stage and instar at study initiation: neonates
- Method of breeding: D. magna was cultured and handled according to the procedures outlined in the ISO-6341
- Source: Kepez Aquaculture Research Institute (Antalya, Turkey)
- Feeding during test: no
ACCLIMATION: not described

Test type:

static

Water media type:

freshwater

Remarks:

dechlorinated tap water

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

EC values were determined after 24, 48, 72 and 96 hours.

Post exposure observation period:

not described

Hardness:

77.5 ± 1.2 mg/L

Test temperature:

21.4 ± 2.3 °C

pH:

7.71 ± 0.49

Dissolved oxygen:

6.39 ± 0.45 mg/L

Conductivity:

217.4 ± 16.95 µS/cm

Nominal and measured concentrations:

Nominal concentrations in the 48 h-test were 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 mg/L.
For nominal concentrations used in the 24, 72 and 96 h test, please refer to section “any other details on materials and methods incl. tables”.
No measurement of the concentration has been carried out.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: not specified
- Material, fill volume: glass, 250 mL
- Volume of solution: 100 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used as dilution water (CaCl2 x H2O 290 mg/L, MgSO4 x 7H2O 120 mg/L, NaHCO3 65 mg/L, KCl 6 mg/L).
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
EFFECT PARAMETERS MEASURED: EC1, EC5, EC10, EC15, EC50, EC85, EC90, EC95, EC99

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 6.03 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 11.63 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Lethargy in the exposure groups
- Observations on body length and weight: not reported
- Other biological observations: Corrosions were observed in the carapax of dead daphnids, and partial ruptures were observed.
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: Daphnids in the higher exposure groups (higher than 10 mg/L) were moving only when the containers were shaken.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
For further details and EC-values, please see section "any other information on results incl. tables".

Results with reference substance (positive control):

not conducted

Reported statistics and error estimates:

All bioassays were run in duplicate and the results are given as mean. EC50 (median effective concentration) values were calculated using a regression line obtained by plotting the concentration (on a logarithmic scale) against the immobilization percentage on a probit scale and the results were evaluated with probit analysis.

Acute toxicity tests indicated that zinc at higher concentrations had a detrimental effect on the survival of Daphnia magna. The calculated 24, 48, 72 and 96 h EC50 value of zinc chloride, using a static bioassay system for D. magna was 11.63, 6.03, 1.17 and 0.67 mg/L, respectively.

Control mortality was zero and controls did not show any behavioural abnormalities.

The results of acute toxicity test for zinc chloride are presented in Table 1 and expressed as median effective concentration (EC50).

In the exposure groups, it was observed that daphnids showed lethargy. Daphnids in the higher exposure groups (higher than 10 mg/L) were moving only when the containers were shaken. Corrosions were observed in the carapax of dead daphnids, and in higher concentrations partial ruptures were observed.

Table 1: EC values (Mean; Min-Max) (mg Zn²⁺/L), 95% confidence limits of zinc chloride for 24, 48, 72 and 96 h on D. magna

Effect concentration	24 h	48 h	72 h	96 h
EC1	4.81 (3.35-5.95)	1.26 (0.58-1.94)	0.53 (0.38-0.64)	0.13 (0.06-0.21)
EC5	6.23 (4.76-7.33)	2.01 (1.11-2.77)	0.67 (0.52-0.77)	0.22 (0.12-0.30)
EC10	7.15 (5.72-8.21)	2.55 (1.57-3.37)	0.76 (0.62-0.86)	0.28 (0.17-0.37)
EC15	7.85 (6.48-8.87)	3.01 (1.97-3.85)	0.82 (0.69-0.92)	0.33 (0.22-0.42)
EC50	11.63 (10.58-12.72)	6.03 (4.96-7.07)	1.17 (1.07-1.27)	0.67 (0.56-0.79)
EC85	17.24 (15.44-20.39)	12.08 (9.99-16.24)	1.66 (1.51-1.93)	1.36 (1.11-1.89)
EC90	18.93 (16.71-23.04)	14.24 (11.48-20.30)	1.81 (1.62-2.15)	1.60 (1.27-2.38)
EC95	21.73 (18.73-27.70)	18.17 (14.01-28.45)	2.05 (1.79-2.53)	2.05 (1.55-3.37)
EC99	28.14 (23.10-39.28)	28.69 (20.16-54.11)	2.58 (2.17-3.45)	3.27 (2.24-6.52)

 

 

Validity criteria fulfilled:

not specified

Remarks:

No daphnids were immobilised in negative controls, but dissolved oxygen concentration was not checked at the end of the test.

Conclusions:

The 48 h EC50 of zinc (Zn2+) in Daphnia magna was 6.03 mg/L.

Executive summary:

In the publication by Ergönül et al., 2012, the toxicity of zinc towards Daphnia magna was examined. The study conditions were similar to OECD guideline 202 with acceptable restrictions: dissolved oxygen concentration, pH and concentration of test substance after test termination are not reported. Zn was added as zinc chloride (ZnCl₂).10 animals per test concentration (duplicates) were used, but EC were determined over a wide range of test item concentrations ranging from 1 – 15 mg/L in the 48-h test group. The EC were determined after 24, 48, 72 and 96 h of exposure.

The calculated 24, 48, 72 and 96 h EC 50 value of zinc, using a static bioassay system for D. magna was 11.63, 6.03, 1.17 and 0.67 mg/L, respectively.

Control mortality was zero and controls did not show any behavioural abnormalities.

In the exposure groups, it was observed that daphnids showed lethargy. Daphnids in the higher exposure groups (higher than 10 mg/L) were moving only when the containers were shaken. Corrosions were observed in the carapax of dead daphnids, and in higher concentrations partial ruptures were observed.

In this study, the EC50 (48 h) of Zn²⁺ was determined to be 6.03 mg/L.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting 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:

1984

Deviations:

yes

Remarks:

No information on dissolved oxygen, temperature during the test,...

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

not described

Vehicle:

no

Details on test solutions:

Control: test medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna K6
- Age at study initiation: < 24 h
- Feeding during test: not explicitly described, but according to OECD202

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Various media were tested.

Test temperature:

Not specified. Media were stored in the test cups at 20 °C.

pH:

Various pH values were tested: 6.0, 6.5, 7.0, 7.5, 8.0. A maximum difference of 0.2 pH units before and after the test was allowed.

Nominal and measured concentrations:

At least six treatments (control and five zinc concentrations) with a maximal difference of one log unit between the lowest and highest zinc concentrations tested.

Details on test conditions:

TEST SYSTEM
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: various media were tested
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED: EC50 (24, 48 h)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.4 µmol/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: at pH 6.0

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11.4 µmol/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: at pH 7.5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

22.7 µmol/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: at pH 8.0

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.93 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: average of results of test at three pH-levels

Details on results:

None reported.

Reported statistics and error estimates:

The zinc speciation in each test medium was calculated using WHAM 5.0.

Validity criteria fulfilled:

not applicable

Conclusions:

The 48 h EC50 of zinc (Zn2+) in Daphnia magna was approximately 0.93 mg/L.

Executive summary:

In the study of Heijerick et al. (2002), the acute zinc toxicity towards Daphnia magna depending on key water chemistry characteristics was examined. The study was carried out according to guideline OECD202, but substantial information was not reported. The test substance was zinc chloride.

The concentration range was described as the maximal difference of one log unit between the lowest and highest zinc concentrations tested. Each treatment was performed with three replicates using 10 organisms per replicate. The number of immobilized juveniles in each cup was recorded after 24 and 48 h.

Three 48 h EC50 have been reported for measurements at pH 6.0, 7.5 and 8.0.

The average 48 h EC50 of Zn²⁺ in Daphnia magna of the three measurements was 0.93 mg/L.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

yes

Remarks:

The authors state that the test was carried out according to OECD guideline 202, but the documentation lacks details.

GLP compliance:

no

Remarks:

GLP compliance was not reported in this publication.

Specific details on test material used for the study:

ZnSO4 was purchased from Sigma-Aldrich (Steinheim, Germany).

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Methanol

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Chemical name of vehicle: methanol, purity > 99%, from Sigma-Aldrich
- Concentration of vehicle in test medium: stock solution 1 mg/mL; maximum concentration of methanol in test medium: 0.001 %
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- common name: water flea
- strain: Daphnia magna
- Age at study initiation (mean and range, SD): < 15 hours
- Stage and instar at study initiation: neonates
- Source: MicroBioTests Inc., Gent, Belgium
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not reported

Test temperature:

22.0 ± 0.5 °C

Nominal and measured concentrations:

Nominal concentrations: 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, 20.00 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: six-well cell culture plates (Cellstar, greiner bio-one, Germany)
- Type: not specified
- Volume of solution: 10 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO standard freshwater, MicroBioTests Inc., Gent, Belgium
OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
EFFECT PARAMETERS MEASURED: EC50 after 48 h exposure
VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.98 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Details on results:

- Other biological observations: internal injuries were observed in immobilized D. magna exposed to ZnSO4

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The observed values of ECx were plotted on a fitted sigmoidal dose-response curve, and were calculated by the Sigmoidal, Sigmoid 3-parameter equation (Sigma-Plot version 12.5, Systat Software, Inc., San Jose, CA, USA). All data are shown as mean ± standard error of mean (SEM).

In phenotypic observation, internal injuries were observed in immobilized D. magna with the ZnSO₄ exposure, but not in the control. The authors assume that the toxicity of ZnSO₄ to D. magna was caused by a direct effect that induces final death through internal absorption.

The adverse effects of ZnSO₄ were shown by a sigmoidal dose-response curve, and were found to be concentration-dependent. After 48 hours of exposure, the EC50 value was 4.89 mg/L, indicating that ZnSO₄ had a highly toxic effect on D. magna.

Validity criteria fulfilled:

not specified

Remarks:

The documentation is not detailed enough to decide whether validity criteria are fulfilled.

Conclusions:

The 48 h EC50 value of zinc sulfate in Daphnia magna was 4.89 mg/L.

Executive summary:

In the publication by Park et al., 2019, the toxicity of zinc sulfate towards Daphnia magna was examined. The study was carried out following OECD guideline 202, Version 2004, according to the authors. The documentation lacks details on dissolved oxygen and pH.

10 animals per test concentration (4 replicates) were used in a static test with six-well plates. ZnSO₄ nominal concentrations were: 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00 and 20.00 mg/L. The EC50 was determined after 48 h of exposure.

Control mortality was zero and no abnormalities in controls were reported. In the ZnSO₄ exposure groups, it was observed that daphnids showed internal injuries.

In this study, the EC50 (48 h) of zinc sulfate was determined to be 4.89 mg/L which corresponds to a Zn²⁺ EC50 (48 h) of 1.98 mg/L.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
ECOSAR v 1.11
2. MODEL (incl. version number)
ECOSAR v 1.11; The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 56-40-6
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For more detailed information please refer to the 'attached justification' section
5. APPLICABILITY DOMAIN
For more detailed information please refer to the 'attached justification' section
6. ADEQUACY OF THE RESULT
For more detailed information please refer to the 'attached justification' section

Qualifier:

no guideline followed

Principles of method if other than guideline:

QSAR prediction of dose descriptors using EPISuite ™, The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012).

GLP compliance:

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

32 749.543 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

Value obtained from a QSAR estimation

Validity criteria fulfilled:

not applicable

Conclusions:

In the present QSAR prediction which was conducted using EPISuite ™, The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012) for glycine, the estiamted LC50 value for glycine was reported to be 32749.543 mg/L. The prediction is reliable because the substance falls into the applicability domain of the model. Moreover, cut-off values that limit the predictions reliability were not exceeded. Based on the presented results glycine does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In the present QSAR prediction conducted with the EPISuite TM; ECOSAR v1.11 software. The program detected two functional groups present in the molecule which are considered to exhibit a more specific mode of toxicity, namely, aliphatic amines and amides. In consideration of the results of all three chemical classes, several LC50 values are estimated. The LC50 value predicted for L-glycine is far above the 100 mg/L limit concentration. Since the substance falls in the applicability domain of the used model and the predicted values are more than 10-fold greater than the water solubility, no toxic effects are expected to occur up to saturation limits.

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

No information on control is provided. Measurement of dissolved oxygen concentration at test termination was not carried out.

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Demineralized (conductivity less than 5 µs cm–1) water was used to prepare stock solution of ZnSO4 x 7H2O immediately before start of tests.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna clone HK
- Age at study initiation (mean and range, SD): < 24 h
- Length at study initiation (length definition, mean, range and SD): mean length 0.813 ± 0.055 mm
- Stage and instar at study initiation: neonates
- Method of breeding: Mothers were kept in groups of 10 animals per 1000 ml of medium at 20 ± 1 °C, with a light-dark regime of 16:8 h and food level of 2 mg C L-1 of Scenedesmus acutus MEYEN.
- Feeding during test: no
ACCLIMATION
- Acclimation period: 3 months
- Acclimation conditions (same as test or not): 40-µm sieve filtered pond water

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

24 and 48 h exposure

Test temperature:

20 ± 1 °C

pH:

7.8 ± 0.2

Dissolved oxygen:

> 7 mg/L

Nominal and measured concentrations:

Nominal concentrations were: 910 to 7822 mg ZnSO4 x 7H2O L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type: not specified
- Material, size: glass, 150 mL
- Volume of solution: 80 mL
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium was prepared according to EN ISO 6341
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: not described
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50 24 h, EC50 48 h

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.254 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.461 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Reported statistics and error estimates:

EC50 (24, 48 h) values and dose-response curves were calculated by non-linear regression using four parameter logistic equation (Motulsky and Christopoulos 2003) by the computer program GraphPad PRISM, version 4.0.

At both exposure times, dose-response curves obtained were sigmoid in shape.

The EC50 (24) value (mean, 95 % Confidence Limits, slope) for D. magna was 4461 µg L-1, range 4025-4944 µg L-1, slope 1.92.

The EC50 (48) value (mean, 95 % Confidence Limits, slope) for D. magna was 2254 µg L-1, range 2254-2587 µg L-1, slope 1.86.

Validity criteria fulfilled:

not applicable

Conclusions:

The 48 h EC50 of zinc (Zn2+) towards Daphnia magna was 2.254 mg/L.

Executive summary:

In the publication by Vesela and Vijverberg (2007), the toxicity of zinc added as zinc sulfate heptahydrate towards Daphnia magna was examined. The study experimental conditions were similar to OECD guideline 202.

Neonates not older than 24 h were used in all tests. Nominal test concentrations of zinc sulfate heptahydrate were 0.910 to 7.822 mg /L. EC50 were determined after 24 and 48 h. Biological observations were not reported.

The 48 h EC50 of Zn²⁺ in Daphnia magna was 2.254 mg/L.

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

According to the authors, acute tests were carried out over 48 hr using standard techniques (OECD, 1981). However, details on experimental conditions are scarce.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna; 6 different genotypes were tested
- Feeding during test: no

Test type:

not specified

Water media type:

freshwater

Remarks:

ASTM hard water

Limit test:

no

Total exposure duration:

48 h

Nominal and measured concentrations:

250-1500 ppb

Details on test conditions:

250-1500 ppb

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Details on results:

No details provided.

Reported statistics and error estimates:

Proportional mortality with concentration was expressed as a normal probability density function for each combination of genotype and toxicant; these, together with EC50s, were estimated using the standard probit method (Finney, 1971).

Table 1: ACUTE EC50 FOR ZINC AS ZINC SULFATE FOR EACH OF THE SIX CLONES TESTED.

Daphnia clone	Zn(II) ppb
A	1831
A-1	755.5
A-2	794.8
B	862.1
E	986.4
S-1	798.1
average	1004.65

1004.65 ppm equal approximately 1.00 mg/L.

Validity criteria fulfilled:

no

Conclusions:

The 48 h EC50 of zinc (Zn2+) in Daphnia magna was approximately 1.00 mg/L.

Executive summary:

In the publication by Baird et al. (1991), six different clones of Daphnia magna were tested for their sensitivity towards zinc. No OECD guideline was followed. The documentation is not detailed enough to allow for a decision on the reliability of the study.

Zinc was added as ZnSO₄. The reported zinc nominal concentrations ranged from approximately 0.25 to 1.50 mg/L. The EC50 was determined after 48 h of exposure.

Effects on controls and abnormalities in the treatment groups are not described.

The 48 h EC50 of Zn²⁺ in six different clones of Daphnia magna clones was 1.00 mg/L in average.

Reference 8

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 zinc monoglycinate sulfate and the source substances zinc sulfate and zinc bisglycinate consist of the Zn2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance zinc monoglycinate sulfate and the source substance zinc bisglycinate.
It is generally accepted that the Zn2+ cation (as measure for dissolved zinc 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 zinc monoglycinate sulfate is a chelate-complex which consists of the divalent zinc ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Zinc monoglycinate sulfate and the source substance zinc sulfate are ionic and consist of the Zn2+ cation and the respective anions. It is generally accepted that the zinc 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, zinc, 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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

3.11 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: calculated for zinc monoglycinate sulfate monohydrate (target substance)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.799 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Zn2+

Basis for effect:

mortality

Remarks on result:

other: Attar and Maly, 1982

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.03 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: Ergönül et al., 2012

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.93 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: Heijerick et al., 2002

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.98 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: Park et al., 2019

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.254 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: Vesela and Vijverberg, 2007

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Zn2+

Basis for effect:

mobility

Remarks on result:

other: Baird et al. (1991)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

32 749.543 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: obtained from QSAR estimation for glycine

Validity criteria fulfilled:

not applicable

Conclusions:

There is no information available on aquatic toxicity of zinc monoglycinate sulfate. However, based on the information available for the constituents glycine and Zn2+, the read-across is based on the hypothesis that the metal ion in combination with amino acids does not exhibit higher ecotoxicity than the metal ion alone. The 48-h EC50 or LC50 for aquatic invertebrates (Daphnia magna) was determined in various studies. The LC50 value chosen in this approach is 0.799 mg Zn2+/L according to Attar and Maly (1982), which is the lowest reported effect concentration.
This corresponds to a 48-h LC50 of 3.11 mg zinc monoglycinate sulfate monohydrate/L in Daphnia magna.

Executive summary:

 There is no information available on aquatic toxicity of zinc monoglycinate sulfate. However, based on the information available for the constituents glycine and Zn²⁺, the read-across is based on the hypothesis that the metal ion in combination with amino acids does not exhibit higher ecotoxicity than the metal ion alone.
 The anions of the target substance are the essential amino acid glycine and sulfate. These anions are not considered as ecotoxicologically relevant at the given concentrations. Instead, Zn²⁺ is the ecotoxicologically critical component.
 The zinc 48-h EC50 or LC50 for aquatic invertebrates (Daphnia magna) was determined in various studies. The LC50 value chosen in this approach is 0.799 mg Zn²⁺/L according to Attar and Maly (1982), for the following reasons: the reported 48-h EC50 values in the most reliable studies (RL2) range from 0.799 to 6.03 mg Zn²⁺/L. The well documented studies by Attar and Maly (1982) and Heijerick et al. (2002) report 48-h values of 0.799 (LC50) and 0.93 mg Zn²⁺/L (EC50), respectively, which are in a very close range. For precautionary reasons, the more conservative value was chosen.
 Apart from effect concentrations, Park et al. (2019) as well as Ergönül et al. (2012) report adverse effects of Zn²⁺ on Daphnia magna like internal injuries, lethargy, corrosion in the carapax and partial ruptures.
 The 48 -h LC50 value chosen in this approach is 0.799 mg Zn²⁺/L.
 This corresponds to a 48-h LC50 of 3.11 mg zinc monoglycinate sulfate monohydrate/L in Daphnia magna.

Description of key information

There is no information available on aquatic toxicity of zinc monoglycinate sulfate. However, based on the information available for the constituents glycine and Zn²⁺, the read-across is based on the hypothesis that the metal ion in combination with amino acids does not exhibit higher ecotoxicity than the metal ion alone.
The anions of the target substance are the essential amino acid glycine and sulfate. These anions are not considered as ecotoxicologically relevant at the given concentrations. Instead, Zn²⁺ is the ecotoxicologically critical component.
The zinc 48-h EC50 or LC50 for aquatic invertebrates (Daphnia magna) was determined in various studies. The LC50 value chosen in this approach is 0.799 mg Zn²⁺/L according to Attar and Maly (1982), for the following reasons: the reported 48-h EC50 values in the most reliable studies (RL2) range from 0.799 to 6.03 mg Zn²⁺/L. The well documented studies by Attar and Maly (1982) and Heijerick et al. (2002) report 48-h values of 0.799 (LC50) and 0.93 mg Zn²⁺/L (EC50), respectively, which are in a very close range. For precautionary reasons, the more conservative value was chosen.
Apart from effect concentrations, Park et al. (2019) as well as Ergönül et al. (2012) report adverse effects of Zn²⁺ on Daphnia magna like internal injuries, lethargy, corrosion in the carapax and partial ruptures.
 The 48 -h LC50 value chosen in this approach is 0.799 mg Zn²⁺/L.
 This corresponds to a 48-h LC50 of 3.11 mg zinc monoglycinate sulfate monohydrate/L in Daphnia magna.

- Attar and Maly, 1982: RL2, no guideline followed, but well documented: The 48 h LC50 of zinc (Zn²⁺) in Daphnia magna was 0.799 mg/L in a flow-through experiment. This corresponds to a 48-h LC50 of 3.11 mg zinc monoglycinate sulfate monohydrate/L in Daphnia magna.

- Ergönül et al., 2012: OECD202, RL2: The 48 h EC50 of zinc (Zn²⁺) in Daphnia magna was 6.03 mg/L. Daphnids in the higher exposure groups (higher than 10 mg/L) showed lethargy and were moving only when the containers were shaken. Corrosions were observed in the carapax of dead daphnids, and in higher concentrations partial ruptures were observed.

- Heijerick et al., 2002: OECD202, RL2: The 48 h EC50 of zinc (Zn²⁺) in Daphnia magna was approximately 0.93 mg/L.

- Park et al., 2019: OECD 202, RL2: The 48 h EC50 value of zinc sulfate in Daphnia magna was 4.89 mg/L which corresponds to an EC50 (48 h) of 1.98 mg Zn²⁺/L. In the ZnSO₄ exposure groups, it was observed that daphnids showed internal injuries.

- Vesela and Vijverberg, 2007: OECD 202, RL2: The 48 h EC50 of zinc (Zn²⁺) towards Daphnia magna was 2.254 mg/L.

- QSAR prediction, RL2: glycine does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

- supporting Baird et al. 1991: RL4, no guideline followed: The 48 h EC50 of zinc (Zn²⁺) in Daphnia magna was approximately 1.00 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

ca. 3.11 mg/L

Additional information

In the publication by Attar and Maly (1982), the toxicity of zinc towards Daphnia magna was examined in a continuous flow apparatus was used for the experiments rather than a static system. Zinc chloride (ZnCI₂) was used in this study. Metal quantities in all experimental tanks were determined daily. The concentrations ranged between 10.6 and 450.9 µg/L. Twenty-three different zinc concentrations were tested. Observations were made every three hours. Mortality was determined by the lack of movement of the second antennules and internal organs in a five second period of observation. No deaths were found in controls in any experiment. The calculated LC50 values of zinc for 36, 48, 60, 72, and 96 hours were reported to be 861.06, 798.94, 420.25, 126.10, and 67.91 µg/L, respectively. Therefore, the LC50 (48 h) of Zn²⁺ to Daphnia magna was 0.799 mg/L in this flow-through experiment.

In the publication by Ergönül et al., 2012, the toxicity of zinc towards Daphnia magna was examined. The study conditions were similar to OECD guideline 202 with acceptable restrictions: dissolved oxygen concentration, pH and concentration of test substance after test termination are not reported. Zn was added as zinc chloride (ZnCl₂).10 animals per test concentration (duplicates) were used, but EC were determined over a wide range of test item concentrations ranging from 1 – 15 mg/L in the 48-h test group. The EC were determined after 24, 48, 72 and 96 h of exposure. The calculated 24, 48, 72 and 96 h EC 50 value of zinc, using a static bioassay system for D. magna was 11.63, 6.03, 1.17 and 0.67 mg/L, respectively. Control mortality was zero and controls did not show any behavioural abnormalities. In the exposure groups, it was observed that daphnids showed lethargy. Daphnids in the higher exposure groups (higher than 10 mg/L) were moving only when the containers were shaken. Corrosions were observed in the carapax of dead daphnids, and in higher concentrations partial ruptures were observed. In this study, the EC50 (48 h) of Zn²⁺was determined to be 6.03 mg/L.

In the study of Heijerick et al. (2002), the acute zinc toxicity towards Daphnia magna depending on key water chemistry characteristics was examined. The study was carried out according to guideline OECD202, but substantial information was not reported. The test substance was zinc chloride. The concentration range was described as the maximal difference of one log unit between the lowest and highest zinc concentrations tested. Each treatment was performed with three replicates using 10 organisms per replicate. The number of immobilized juveniles in each cup was recorded after 24 and 48 h. Three 48 h EC50 have been reported for measurements at pH 6.0, 7.5 and 8.0. The average 48 h EC50 of Zn²⁺ in Daphnia magna of the three measurements was 0.93 mg/L.

In the publication by Park et al., 2019, the toxicity of zinc sulfate towards Daphnia magna was examined. The study was carried out following OECD guideline 202, Version 2004, according to the authors. The documentation lacks details on dissolved oxygen and pH. 10 animals per test concentration (4 replicates) were used in a static test with six-well plates. ZnSO₄ nominal concentrations were: 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00 and 20.00 mg/L. The EC50 was determined after 48 h of exposure. Control mortality was zero and no abnormalities in controls were reported. In the ZnSO₄ exposure groups, it was observed that daphnids showed internal injuries. In this study, the EC50 (48 h) of zinc sulfate was determined to be 4.89 mg/L which corresponds to a Zn²⁺EC50 (48 h) of 1.98 mg/L.

In the publication by Vesela and Vijverberg (2007), the toxicity of zinc added as zinc sulfate heptahydrate towards Daphnia magna was examined. The study experimental conditions were similar to OECD guideline 202, but there is a lack of information on control results so that it cannot be decided whether validity criteria are fulfilled. Neonates not older than 24 h were used in all tests. Nominal test concentrations of zinc sulfate heptahydrate were 0.910 to 7.822 mg /L. EC50 were determined after 24 and 48 h. Biological observations were not reported.The 48 h EC50 of Zn²⁺ in Daphnia magna was 2.254 mg/L.

Glycine: In the present QSAR prediction which was conducted using EPISuite ™, The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012) for glycine, the estiamted LC50 value for glycine was reported to be 32749.543 mg/L. The prediction is reliable because the substance falls into the applicability domain of the model. Moreover, cut-off values that limit the predictions reliability were not exceeded. Based on the presented results glycine does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

In the publication by Baird et al. (1991), six different clones of Daphnia magna were tested for their sensitivity towards zinc. No OECD guideline was followed. The documentation is not detailed enough to allow for a decision on the reliability of the study. Zinc was added as ZnSO₄. The reported zinc nominal concentrations ranged from approximately 0.25 to 1.50 mg/L. The EC50 was determined after 48 h of exposure. Effects on controls and abnormalities in the treatment groups are not described. The 48 h EC50 of Zn²⁺ in six different clones of Daphnia magna clones was 1.00 mg/L in average.