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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2016 to 11 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]

Version / remarks:

Second Edition 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 29763-2013, Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test [S].

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 27861-2011, Chemicals Fish Acute toxicity test [S]. Beijing: Standards Press of China

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures [S].

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ/T 153-2004. The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal concentrations of 34.9, 50.2, 72.3, 104 and 150 mg/L
- Sampling method: The actual concentrations of the test substance in all test substance groups were determined at 0 h, 72 h (new solution) and 24, 96 (old solution) by HPLC, and the blank control was measured at 0 h (new solution) and 24 h (old solution).
- Sample storage conditions before analysis: The samples were analysed immediately or stored after dilution at 2 to 8 °C protected from light. In this case, analysis was carried out within 4 days.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
0.314, 0.452, 0.651, 0.936 and 1.350 g of test material were respectively obtained in five flasks and dissolved with a small amount of test water with ultrasonication treatment for 15 minutes, then the test solutions were respectively transferred into five 10 L-tanks and diluted with test water to 9000 mL, and the nominal concentrations of 34.9, 50.2, 72.3, 104 and 150 mg/L test solutions were obtained. The test solutions were stirred well and used in the test directly.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Rare Minnow

ACCLIMATION
- Acclimation period: Before the start of the test, Rare Minnow (Gobiocypris rarus) were acclimated for 183 days.
- Acclimation conditions: domestication conditions (water quality: dechlorinated tap water, temperature: 23 ± 2 °C, photoperiod: 14h light / 10h dark). The dissolved oxygen concentration during acclimation was kept above 80 % of the air saturation value.
- Feeding frequency during acclimation: The fish were fed at least three times per week or workdays until 24 hours prior to test start.
- Health during acclimation: The mortality was 0 % of the population for 7 days before the start of the test, which met the requirement of mortalities being less than 5 % of the population.

FEEDING DURING TEST: No, the fish were not fed during exposure.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

110 mg/L (expressed as CaCO3)

Test temperature:

23.1 to 24.0 °C

pH:

The test was performed without adjustment of pH and the pH was in the range of 7.17 to 8.14 during the test period.

Dissolved oxygen:

93 % in the test water. The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test were kept at not less than 60 % of the air saturation value.

Nominal and measured concentrations:

Nominal: 34.9, 50.2, 72.3, 104 and 150 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 L tank (25 x 20 x 20 cm)
- Material, size, headspace, fill volume: 9 L/vessel
- Aeration: No
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.128 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water was used in the test. The total hardness of test water was 110 mg/L (expressed as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: The test was performed without adjustment of pH and the pH was in the range of 7.17 to 8.14 during the test period.
- Photoperiod: Artificial light of white fluorescent lamp was used. The photoperiod was 14h light / 10h darkness.
- Light intensity: At the beginning and the end of the test, the intensities of illumination of test environment were 316 lx and 308 lx on average, respectively.
- Type of test: The measured concentration of test substance in test solution could be maintained within ± 20 % of the nominal value, but the dissolved oxygen concentration could not be kept above 60 % of the air saturation value for the criterion of the test validity. Consequently, the definitive test was performed as semi-static at 24 h intervals.

OBSERVATIONS AND MEASUREMENTS
- Measurement of light illumination: The intensity of illumination was measured at the beginning and end of the test.
- Observation of test solutions appearance: The changes of colour and appearance of the test solutions were recorded daily.
- Observation of test organisms: Mortalities and visible abnormalities of the test fish were recorded at 3, 24, 48, 72 and 96 h after the start of the test during test period. The dead fish were removed as soon as they were found. A fish is considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction.
- Measurement of temperature, pH and dissolved oxygen of test solution: The temperature, pH and dissolved oxygen of each test group were measured at 0h, 96h and 24h, 48h, 72h (before and after renewal) after the start of the test. Measurements of pH were determined with a portable pH meter. Measurements of temperature and dissolved oxygen were determined with an oxygen meter. The temperature of test water was continuously monitored during the test exposure by dual thermometer.
- Body weight and total length of test fish: The body weight and total length of 7 test fish in blank control were measured at the end of the test.

TEST CONCENTRATIONS
- Nominal concentrations:34.9, 50.2, 72.3, 104 and 150 mg/L
- Range finding study: Performed under non-GLP conditions on 21 March 2016.
- Test concentrations: 0, 9.77, 31.2 and 100 mg/L
- Results used to determine the conditions for the definitive study: The range-finding test showed that the 96h cumulative mortalities of test fish in the blank control and the test substance groups of 9.77, 31.2 and 100mg/L were 0, 0, 0 and 80 %, respectively.
Based on the results of the range-finding test and stability test, the definitive test was conducted as semi-static test at 24h intervals. Five nominal concentrations of 34.9, 50.2, 72.3, 104 and 150 mg/L test substance groups and a blank control group were selected.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limits of 35.0 - 54.1 mg/L.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

< 34.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

72.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

71.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limits of 53.2 - 95.2 mg/L.

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

48.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limits of 42.7 - 56.1 mg/L.

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

46.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limits of 41.0 - 52.6 mg/L

Details on results:

RESULTS
INTENSITY OF ILLUMINATION
-At the start of the test, the intensities of illumination of test environment were 310, 298 and 341 lx (316 lx on average).
-At the end of the test, the intensities of illumination of test environment were 293, 305 and 325 lx (308 lx on average).

OBSERVATION OF TEST MATERIAL SOLUTIONS
The blank control solution was observed to be clear and colourless during the whole test period. The new solutions of substance solutions were clear and colourless after being freshly prepared, but before each renewal and at the end of the test, the old solutions were observed to be slightly turbid before renewal.

MORTALITY
-Cumulative mortalities of each group at 3, 24, 48, 72 and 96 hrs after the start of the test are shown in Table 1.
-At the end of the test, the cumulative mortalities of the blank control were 0 %, which met the validity criterion. The cumulative mortalities of 34.9, 50.2, 72.3, 104 and 150 mg/L test groups were respectively 14, 71, 100, 100 and 100 % at the end of the test.

OBSERVED ABNORMAL RESPONSE
The abnormal responses observations of test fish during the test are shown in Table 2. No abnormal responses of test fish were observed in the blank control during the whole test. At 3 h after the start of the test, all test fish of the substance were observed to be normal. At 24 h, some fish died in the test groups of 50.2, 72.3, 104 and 150 mg/L, and some fish observed to be reduction of activity, overturn and loss of equilibrium, immobilised at the bottom and swimming at the bottom. At 48 h, all fish died in the test groups of 72.3, 104 and 150 mg/L. From 48 to 96 h, the fish alive in the 50.2 mg/L test group were performed reduction of activity. From the test period of 3 to 72h, all fish were normal in 34.9 mg/L test group, but one fish died and one fish overturned and showed loss of equilibrium at the end of the test.

TEMPERATURE, PH AND DISSOLVED OXYGEN
During the test, the measured pH values and dissolved oxygen ranged from 7.17 to 8.14 and from 65 to 92 %, respectively. The temperature of test environment ranged from 23.1 to 24.0 °C. The dissolved oxygen concentrations were kept above 60 % of the air saturation value, which met the criterion for the validity of the test.

BODY WEIGHT AND TOTAL LENGTH OF FISH
The fish’s body weight and total length of the blank control were measured at the end of the test. The average and standard deviation of body weight and total length of test fish were 0.165 ± 0.013 g and 27.39 ± 1.12 mm, respectively.

MEDIAN LETHAL CONCENTRATION (LC50) AND THE 95 % CONFIDENCE LIMITS
At the end of the test, the 96h-LC50 of test substance to Rare Minnow (Gobiocypris rarus) was 44.0 mg/L and the 95 % confidence limits ranged from 35.0 to 54.1mg/L (based on the nominal concentration).

Results with reference substance (positive control):

The sensitivity test of reference substance K2Cr2O7 (performed on March 31, 2016) showed that, the 24 h LC50 of K2Cr2O7 to Rare Minnow (Gobiocypris rarus) was 476 mg/L and the 95 % confidence limits was in the range of 368 to 657 mg/L.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality

Nominal concentration (mg/L)	Number of fish	Cumulative mortality (%)
		3 h	24 h	48 h	72 h	96 h
Blank control	7	0	0	0	0	0
34.9	7	0	0	0	0	14
50.2	7	0	29	57	71	71
72.3	7	0	57	100	100	100
104	7	0	86	100	100	100
150	7	0	86	100	100	100

Table 2: Observed abnormal response

Nominal concentration (mg/L)	Result of observation (Left column: Number of affected fish / Total survival number, Right column: Symptom detail)
	3 h		24 h		48 h		72 h		96 h
Blank control	0/7	N	0/7	N	0/7	N	0/10	N	0/7	N
34.9	0/7	N	0/7	N	0/7	N	0/7	N	1/6	OLE(1)
50.2	0/7	N	3/5	RA(2) AS(1)	3/3	RA(3)	2/2	RA(2)	2/2	RA(2)
72.3	0/7	N	3/3	OLE(3)	-	-	-	-	-	-
104	0/7	N	1/1	IMM(1)	-	-	-	-	-	-
150	0/7	N	1/1	IMM(1)	-	-	-	-	-	-

N: Normal, —: All fish died, RA: Reduction of activity, IMM: Immobilised at the bottom, AS: Swimming at the surface, OLE: Overturn and loss of equilibrium.

Validity of the Test

-The mortality of the control group was 0 % at the end of the test, which met the validity criterion that the mortality should be less than 10 %.

-The dissolved oxygen concentration in the test solutions ranged from 65 to 92 % of the air saturation value throughout the test, which met the criterion for the validity of the test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96 h LC50 of test material to Rare Minnow (Gobiocypris rarus) was 44.0 mg/L and the 95 % confidence limits were 35.0 to 54.1mg/L (based on the nominal concentration).

Executive summary:

The potential of the test material to cause acute toxicity to fish was determined in accordance with the standardised guidelines OECD 203 and other Chinese guidelines, under GLP conditions using Rare Minnow (Gobiocypris rarus) fish in a definitive test under semi-static test at 24 hour intervals.

The range-finding test showed that the 96h cumulative mortalities of test fish in the blank control and the test substance groups of 9.77, 31.2 and 100 mg/L were 0, 0, 0 and 80 %, respectively. Based on these results the definitive test was conducted as semi-static test at 24h intervals. Five nominal concentrations of 34.9, 50.2, 72.3, 104 and 150 mg/L test substance groups and a blank control group were selected. Actual concentrations were measured using HPLC.

The cumulative mortalities of 34.9, 50.2, 72.3, 104 and 150 mg/L test groups were respectively 14, 71, 100, 100 and 100 % at the end of the test. At 3 h after the start of the test, all test fish of the substance were observed to be normal. At 24 h, some fish died in the test groups of 50.2, 72.3, 104 and 150 mg/L, and some fish observed to be reduction of activity, overturn and loss of equilibrium, immobilised at the bottom and swimming at the bottom. At 48 h, all fish died in the test groups of 72.3, 104 and 150 mg/L. From 48 to 96 h, the fish alive in the 50.2 mg/L test group were performed reduction of activity. From the test period of 3 to 72h, all fish were normal in 34.9 mg/L test group, but one fish died and one fish overturned and showed loss of equilibrium at the end of the test.

The mortality of the control group was 0 % at the end of the test, which met the validity criterion that the mortality should be less than 10 %. The dissolved oxygen concentration in the test solutions ranged from 65 to 92 % of the air saturation value throughout the test, which met the criterion for the validity of the test. Potassium dichromate was used as a positive control reference substance to demonstrate the acceptability of the conditions of the assay.

Under the conditions of this study, the 96h-LC50 of the test material to Rare Minnow (Gobiocypris rarus) was 44.0 mg/L and the 95 % confidence limits ranged from 35.0 to 54.1 mg/L (based on the nominal concentration).

Description of key information

Under the conditions of this study, the 96h-LC50 of the test material to Rare Minnow (Gobiocypris rarus) was 44.0 mg/L and the 95 % confidence limits ranged from 35.0 to 54.1 mg/L (based on the nominal concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

44 mg/L

Additional information

The potential of the test material to cause acute toxicity to fish was determined in accordance with the standardised guidelines OECD 203 and other Chinese guidelines, under GLP conditions using Rare Minnow (Gobiocypris rarus) fish in a definitive test under semi-static test at 24 hour intervals. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The range-finding test showed that the 96h cumulative mortalities of test fish in the blank control and the test substance groups of 9.77, 31.2 and 100mg/L were 0, 0, 0 and 80 %, respectively. Based on these results the definitive test was conducted as semi-static test at 24h intervals. Five nominal concentrations of 34.9, 50.2, 72.3, 104 and 150 mg/L test substance groups and a blank control group were selected. Concentrations were measured using HPLC in an analytical method.

The cumulative mortalities of 34.9, 50.2, 72.3, 104 and 150 mg/L test groups were respectively 14, 71, 100, 100 and 100 % at the end of the test. At 3 h after the start of the test, all test fish of the substance were observed to be normal. At 24 h, some fish died in the test groups of 50.2, 72.3, 104 and 150 mg/L, and some fish observed to be reduction of activity, overturn and loss of equilibrium, immobilised at the bottom and swimming at the bottom. At 48 h, all fish died in the test groups of 72.3, 104 and 150 mg/L. From 48 to 96 h, the fish alive in the 50.2 mg/L test group were performed reduction of activity. From the test period of 3 to 72h, all fish were normal in 34.9 mg/L test group, but one fish died and one fish overturned and showed loss of equilibrium at the end of the test.

The mortality of the control group was 0 % at the end of the test, which met the validity criterion that the mortality should be less than 10 %. The dissolved oxygen concentration in the test solutions ranged from 65 to 92 % of the air saturation value throughout the test, which met the criterion for the validity of the test. Potassium dichromate was used as a positive control reference substance to demonstrate the acceptability of the conditions of the assay.

Under the conditions of this study, the 96h-LC50 of the test material to Rare Minnow (Gobiocypris rarus) was 44.0 mg/L and the 95 % confidence limits ranged from 35.0 to 54.1mg/L (based on the nominal concentration).