Bis(5-oxo-L-prolinato-N1,O2)zinc

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

Study period:

14 November 2016 to 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines

Version / remarks:

The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).
JMAFF 2-7-2-1 The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test material concentrations were sampled.
- Sampling method: Analytical measurements were performed from the control and at the applied test concentration levels at the beginning and at the end of the experiment. The samples were analyzed by HPLC-UV method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
-Method: A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test material, mixed into the test medium (ISO Medium) using ultrasonic bath (approximately 3 minutes). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the experiment).
-Untreated Control: The dilution water (ISO-medium) was used without addition of the test material.
-Dilution water: Reconstituted water was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
The medium was prepared with the following:
25 mL of 11.76 g/L CaCl2x2H2O
25 mL of 4.93 g/L MgSO4x7H2O
25 mL of 0.23 g/L KCl
25 mL of 2.59 g/L NaHCO3
-Reference Control: For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age of parental stock: They were less than 24 h old at the beginning of the test.
- The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

246 mg/L (as CaCO3).

Test temperature:

19.8 – 20.1 °C

pH:

6.98 – 7.30

Dissolved oxygen:

8.5 – 9.0 mg/L.

Nominal and measured concentrations:

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25, 50, and 100 mg/L.
The corresponding measured geometric mean test material concentrations were 6.85; 13.89; 27.09; 54.80 and 112.37 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Type: Open
- Material, size, headspace, fill volume: At least 5 mL/animal
- No. of organisms per vessel: 5 animals per replicate
- No. of vessels per concentration: 4 replicates
- No. of vessels per vehicle control: 5 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the experiment in each test vessel and was in the range of 6.98 to 7.30.
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.
- Temperature: The water temperature was measured at the start of the experiment and in 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 19.8 – 20.1 °C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.5 and 20.3 °C.

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under static conditions.
During the formulation procedure the stock solution was prepared by the similar method to the main study. The test solutions were prepared by appropriate dilution of the stock solution.
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. Significant immobility was observed during the preliminary concentration range-finding test and as a result five test concentrations in a geometric series (factor 2.0) and one control group were tested in the main experiment under static conditions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

21.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

37.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

IMMOBILISATION
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour. The number and percentage of immobilised animals can be seen in Table 1.
In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
Even if only biological significance could be observed in the test material treated group of 12.5 mg/L (nominal) and it is not statistically significantly different from the control, the observed effect (15 %) is not negligible (due to that the accepted control immobilization is 10 %). Therefore the NOEC was determined as 6.25 mg/L (nominal) and the LOEC could be determined as the highest concentration level of 12.5 mg/L (nominal).
As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, the biological results are based on the nominal test item concentrations.

Results with reference substance (positive control):

The last study perfomed with the reference material had the following results: 24h EC50 was 0.93 mg/L, (95 % confidence limits: 0.86 – 0.99 mg/L).

Reported statistics and error estimates:

The 24 and 48 hour EC50 values of the test material with their 95 % confidence limits were calculated using Probit analysis by TOXSTAT software. The 48 hours EC100 values of the test material were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 1: The number and percentage of immobilised animals

Nominal concentration (mg/L)	Number of treated animals	Immobilised animals
		24 hours		48 hours
		number	percent	number	percent
Control	20	0	0	0	0
6.25	20	1	5	1	5
12.5	20	3	15**	3	15**
25	20	8	40*	15	75*
50	20	8	40*	16	80*
100	20	19	95*	20	100*

*= statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

**= biologically significantly different compared to the control values

Validity

-There was no immobiliSation in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.

-All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48h EC50 was 21.14 mg/L, the 48h NOEC was 6.25 mg/L and the 48h LOEC was 12.5 mg/L, based on nominal concentrations.

Executive summary:

The acute toxicity to Daphnia magna was investigated in a GLP study in accordance to the standardised guidelines OECD 202, EPA OCSPP 850.1010, EU method C.2 and Japanese guidelines

Acute toxicity of the test material on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a static test system.

Because significant immobility was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25, 50, and 100 mg/L.

The test concentrations were analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test material concentrations were: 6.85; 13.89; 27.09; 54.80 and 112.37 mg/L.

As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentrations and for the control as well.

The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.

For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. Furthermore values were biologically significantly different compared to the control were also considered.

All validity criteria were met during this study.

Under the conditions of the study, the 48h EC50 was 21.14 mg/L, the 48h NOEC was 6.25 mg/L and the 48h LOEC was 12.5 mg/L, based on nominal concentrations.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 July 2015 to 17 July 2015

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:

no

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Dilution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
-The test material was dissolved in the dilution water to prepare a stock solution. The stock solution was diluted in the dilution water to prepare the test solution for each concentration group. The dilution water was used as it was.
-Concentrations were corrected to the purity of the test material (89.7 %)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Age: Female juveniles less than 24 hours old.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 1 °C

Dissolved oxygen:

3 mg/L (not aerated)

Nominal and measured concentrations:

0.050, 0.19, 0.71, 2.7 and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL/vessel
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentratio: 4
- No. of vessels per control: 4

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

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 800 lux or less

EFFECT PARAMETERS MEASURED: Daphnids were observed for immobilisation at 24 and 48 hours of exposure. Individuals unable to swim within 15 seconds after gentle agitation of the test vessel were considered immobile.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- A range finding test was performed but data was not included in the report. The concentrations for the definitive test were determined as a result of the range finder.
-The separation factor for the definitive test was larger than 2.2 because effect of the test material was observed on Daphnids in a wide concentration range in the range-finding test. The separation factor was 3.76.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits of 1.69 - 3.49 mg/L.

Reported statistics and error estimates:

Calculated by probit method.

Table 1: The cumulative numbers and percentages of immobilised daphnids

Concentration (mg/L)	Cumulative number of Immobilised Daphnia (Percent immobility)
	24 hours	48 hours
Control	0 (0)	0 (0)
0.050	0 (0)	0 (0)
0.19	0 (0)	0 (0)
0.71	0 (0)	2 (10)
2.7	3 (15)	9 (45)
10	10 (50)	20 (100)

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study the 48 hour EC50 is 2.43 mg/L (95 % confidence limits 1.69 to 3.49 mg/L).

Executive summary:

The potential of the test material to cause short term toxicity to aquatic invertebrates was determined in accordance with the standardised guideline OECD 202 using a Daphnia magna immobilisation test.

Following from the results of a range-finding test 20 dapnids per test group were exposed to the following concentrations of test material for 48 hours: 0.050, 0.19, 0.71, 2.7 and 10 mg/L. Daphnids were observed for immobilisation after 24 and 48 hours of exposure to the test material.

Under the conditions of the study the 48 hour EC50 is 2.43 mg/L (95 % confidence limits 1.69 to 3.49 mg/L).

Description of key information

Under the conditions of the study, the 48h EC50 was 21.14 mg/L, the 48h NOEC was 6.25 mg/L and the 48h LOEC was 12.5 mg/L, based on nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

21.14 mg/L

Additional information

Key Study: Sipos (2016)

The acute toxicity to Daphnia magna was investigated in a GLP study in accordance to the standardised guidelines OECD 202, EPA OCSPP 850.1010, EU method C.2 and Japanese guidelines. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Acute toxicity of the test material on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a static test system.

Because significant immobility was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25, 50, and 100 mg/L.

The test concentrations were analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test material concentrations were: 6.85; 13.89; 27.09; 54.80 and 112.37 mg/L.

As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentrations and for the control as well.

The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.

For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. Furthermore values were biologically significantly different compared to the control were also considered.

All validity criteria were met during this study.

Under the conditions of the study, the 48h EC50 was 21.14 mg/L, the 48h NOEC was 6.25 mg/L and the 48h LOEC was 12.5 mg/L, based on nominal concentrations.

Supporting Study: Yoshimura (2015)

The potential of the test material to cause short term toxicity to aquatic invertebrates was determined in accordance with the standardised guideline OECD 202 using a Daphnia magna immobilisation test. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Following from the results of a range-finding test 20 dapnids per test group were exposed to the following concentrations of test material for 48 hours: 0.050, 0.19, 0.71, 2.7 and 10 mg/L. Daphnids were observed for immobilisation after 24 and 48 hours of exposure to the test material.

Under the conditions of the study the 48 hour EC50 is 2.43 mg/L (95 % confidence limits 1.69 to 3.49 mg/L).