[No public or meaningful name is available]

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:

14-18 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 203. All validity criteria were fulfilled

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The Netherlands GLP Compliance Programme (inspected on 28 February - 7 March 2014 / signed on 30 April 2014)

Specific details on test material used for the study:

Phys-Chem Properties:
Water Solubility: Well soluble in water (481 mg/L in 2 days experiment; 875 mg/L in 3 days experiment)
Partition Coefficient: log Pow=3.8
Vapour Pressure: 5.8 Pa at 20 °C, 11 Pa at 25 °C

Analytical monitoring:

yes

Details on sampling:

The concentrations of test substance of test media were determined by GC; samples were taken for analysis from the freshly prepared solutions at 0 and 72 hours, and the spent solutions at 24 and 96 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: According to the solubility in water of the test item, test item was added directly into test water to prepare the test solution. The weight of test item was calculated according to the volume of bottles, the test item of 0.03298-0.03488, 0.04949-0.05195, 0.07351-0.08319, 0.11006-0.12355 and 0.16519-0.18853 g were weighted and dissolved in 5 glass bottles (with a stopper) respectively. Test water of about 11 L was filled into the bottles respectively, and then be stirred by magnetic stirrer (400 - 450 rpm) for about 2 hours to get the test solution.
- Controls: A concurrent blank control was included in parallel.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Batch: Zebra fish 20141209
- Strain: AB strain.
- Source: Hunter Biotech
- Length at study initiatio: 2.0 ± 1.0 cm
- Weight at study initiation: Should meet the requirement that test loading is <1.0 g/L

ACCLIMATION
- Feeding frequency during acclimation: Brine shrimp twice on workday and once on weekend which was discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation: There were no any sign of disease or mortalities maintained in-house.

FEEDING DURING TEST: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

136 mg/L (as CaCO3) (met the criteria of 10-250 mg/L).

Test temperature:

23 ± 2 °C (21.07-24.93 °C)

pH:

6.89-8.35

Dissolved oxygen:

77.1-106.3% of the air saturation value (ASV)

Salinity:

None

Nominal and measured concentrations:

Nominal concentrations: 3.0, 4.5, 6.7, 10 and 15 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Test was conducted under a closed system using glass bottles (having a stopper made from an inert material, filled with water to reduce headspace (ca 11 liters volume)).
- Type: Closed
- Aeration: Test water was aerated for at least 24 hours to get the dissolved oxygen saturated.
- Renewal rate of test solution: Test was performed with renewal of the test solutions every 24 hours.
- No. of organisms per test group: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Loading: <1.0 g fish/L water
- Ten fish were put into freshly prepared solutions at the beginning of the test and then transferred to newly prepared solutions after 24 hours.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test water used for the aquatic test was ground water treated with aquapro pure water system.

OTHER TEST CONDITIONS
- Photoperiod: 12 hours darkness: 12 hours light

EFFECT PARAMETERS MEASURED
Observations: The fish in the test and control groups were observed daily. Toxic signs and mortality were recorded at 2, 4, 24, 48, 72 and 96 hours during exposure. Fish was considered dead if there was no visible movement (e.g. gill movements) and no reaction when touching of the caudal peduncle. Dead fish were removed when observed, and mortalities were recorded. Any of the visible abnormalities (e.g. pigmentation, lethargy, loss of equilibrium, etc.) were recorded. During 96 hours test period, the dissolved oxygen and pH of fresh media and of expired media of test were determined daily. And the temperature was monitored by automated temperature monitoring system and recorded at 1 hour interval.
Euthanasia of tested fish: After 96 hours of exposure, all of the surviving fish were euthanized. The method of euthanasia was to put fish into a 0.5% water solution of ethylene glycol monophenyl ether.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP preliminary test, the concentrations of 2, 3, 5, 10, 15 and 100 mg/L were conducted in the preliminary test. The test solution was prepared by adding test item into test water and stirring for 2 hours or 24 hours. 5 fish were used for each concentration.
- Results used to determine the conditions for the definitive study: The results of a preliminary test showed that the 96-hour LC50 was located between 3 - 15 mg/L (nominal concentration). Based on the results of preliminary test, the definitive test was carried out as a semi-static-exposure test with 5 test item concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L.A concurrent blank control was included in parallel.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

11.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limit of 10.20-12.51 mg/L

Details on results:

During the 96-hour exposure period, there was no death and visible toxic sign observed in the vessels of control group, 3.0 and 4.5 mg/L group.
There was lethargy observed in the vessel of 6.7 mg/L, but no death was observed. There were lethargies, discolorations, hemorrhaging, erratic-swimming and lying in the side observed in the vessels of 10 and 15 mg/L. There were only 2 deaths in 10 mg/L, but all test fish in 15 mg/L were dead before the end of the test.

Results with reference substance (positive control):

The reference substance test of potassium dichromate for this batch fish is conducted from July 06, 2015 to July 07, 2015. The LC50 (24 hour) result of reference substance test to zebra fish is 321.19 mg/L, with 95% confidence limits of 283.55-363 .84 mg/L.

Reported statistics and error estimates:

Trimmed Spearman-Karber (TSK) method (EPA, Version 1.5) was used to derive the 96 h-LC50 value and the 95% confidence limit. And the LC50 was calculated with nominal concentrations.

Sublethal observations / clinical signs:

Table 6.1.1/1: Cumulative Mortality in the Definitive Test

 

Nominal Concentration (mg/L)	Determined Concentration (mg/L)	Initial Population	Time (h)	Cumulative Mortality	Abnormalities
Control	-	10	2	0	10 No poisoning sign
			4	0	10 No poisoning sign
			24	0	10 No poisoning sign
			48	0	10 No poisoning sign
			72	0	10 No poisoning sign
			96	0	10 No poisoning sign
3.0	2.79	10	2	0	10 No poisoning sign
			4	0	10 No poisoning sign
			24	0	10 No poisoning sign
			48	0	10 No poisoning sign
			72	0	10 No poisoning sign
			96	0	10 No poisoning sign
4.5	4.42	10	2	0	10 No poisoning sign
			4	0	10 No poisoning sign
			24	0	10 No poisoning sign
			48	0	10 No poisoning sign
			72	0	10 No poisoning sign
			96	0	10 No poisoning sign
6.7	6.68	10	2	0	10 No poisoning sign
			4	0	10 No poisoning sign
			24	0	10 No poisoning sign
			48	0	10 No poisoning sign
			72	0	10 No poisoning sign
			96	0	10 lethargy
10	10.1	10	2	0	10 lethargy
			4	0	10 lethargy
			24	0	10 lethargy and 8 discoloration
			48	0	2 erratic swimming, 8 discoloration and lethargy
			72	0	2 erratic swimming, 8 discoloration and lethargy
			96	2	8 discoloration and lethargy
15	15.4	10	2	0	2 erratic swimming and discoloration; 8 lethargy and discoloration
			4	0	4 erratic swimming and discoloration; 5 lethargy and discoloration 1 hemorrhaging and discoloration
			24	0	2 lethargy and discoloration 8 lying in the side
			48	1	9 lying in the side
			72	10	-

 

Calculation of LC50

Basing on the analytically confirmed nominal concentrations, the 96-hour mortality data of the test was calculated by Trimmed Spearman-Karber (TSK) method (EPA, Version 1.5) to derive the LC50 value and the 95% confidence limit. The 96-hour LC50 of test substance to zebra fish was 11.30 mg/L with the 95% confidence limits of 10.20-12.51 mg/L.  

Verification of test concentration

The results showed that for the nominal concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L, the mean determined concentrations were 2.86, 4.45, 6.75, 10.0 and 15.3 mg/L in the fresh solutions and 2.72, 4.39,6.61,10.1 and 15.4 mg/L in the spent solutions. The overall determined concentrations were 2.79, 4.42, 6.68, 10.1 and 15.4 mg/L, which were 92.9, 98.3, 99.7, 101 and 102% of nominal concentrations, respectively.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 96-hour LC50 of test substance to zebra fish was 11.30 mg/L with the 95% confidence limit of 10.20-12.51 mg/L and based on analytically confirmed nominal concentrations.

Executive summary:

The acute toxicity of the test item to zebra fish (Danio rerio) was conducted according to OECD guideline 203 and in compliance with GLP.

The test was performed with a semi-static water renewal system (solutions were replaced every 24 hours) with the nominal test item concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L. Duration of the test was 96 hours. 10 fish were used in each of the test groups and the control group. Water quality parameters temperature, pH-value and oxygen-saturation measured were determined to be within the acceptable limits. The determination of the test concentrations of test item was carried out via GC-MS.

 

During the 96-hour exposure period, there was no death and visible toxic sign observed in the vessels of control group, 3.0 and 4.5 mg/L group. There was lethargy observed in the vessel of 6.7 mg/L, but no death was observed. There were lethargies, discolorations, hemorrhaging, erratic-swimming and lying in the side observed in the vessels of 10 and 15 mg/L. There were only 2 deaths in 10 mg/L, but all test fish in 15 mg/L were dead before the end of the test.

 

The results showed that for the nominal concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L, the mean determined concentrations were 2.86, 4.45, 6.75, 10.0 and 15.3 mg/L in the fresh solutions and 2.72, 4.39, 6.61, 10.1 and 15.4 mg/L in the spent solutions. The overall determined concentrations were 2.79, 4.42, 6.68, 10.1 and 15.4 mg/L, which were 92.9, 98.3, 99.7, 101 and 102% of nominal concentrations, respectively.

 

Based on the analytically confirmed nominal concentrations, the 96-hour mortality data of the test was calculated by Trimmed Spearman-Karber (TSK) method (EPA, Version 1.5) to derive the LC50 value and the 95% confidence limit.

 

Under the test conditions, the 96-hour LC50 of test substance to zebra fish was 11.30 mg/L with the 95% confidence limit of 10.20-12.51 mg/L and based on analytically confirmed nominal concentrations.

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LC50 (Danio rerio) = 11.3 mg/L (95% CI: 10.2 -12.51 mg/L) based on analytically confirmed nominal concentrations

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

11.3 mg/L

Additional information

One key study is available (SYRICI, 2015) to assess the 96h acute toxicity of the registered substance to zebra fish (Danio rerio) under semi-static conditions according to OECD guideline 203 with GLP compliance.

The test was performed with a semi-static water renewal system (solutions were replaced every 24 hours) with the nominal test item concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L.

Duration of the test was 96 hours. 10 fish were used in each of the test groups and the control group. Water quality parameters temperature, pH-value and oxygen-saturation measured were determined to be within the acceptable limits. The determination of the test concentrations of test item was carried out via GC-MS. A semi-static test sytem was used and the test solutions were analyzed during the study. Samples were taken for analysis from the freshly prepared solutions at 0 and 72h, and the spent solutions at 24 and 96 hours.

 The results showed that for the nominal concentrations of 3.0, 4.5, 6.7, 10 and 15 mg/L, the mean determined concentrations were 2.86, 4.45, 6.75, 10.0 and 15.3 mg/L in the fresh solutions and 2.72, 4.39, 6.61, 10.1 and 15.4 mg/L in the spent solutions. The overall determined concentrations were 2.79, 4.42, 6.68, 10.1 and 15.4 mg/L, which were 92.9, 98.3, 99.7, 101 and 102% of nominal concentrations, respectively.

Based on the analytically confirmed nominal concentrations, the 96-hour mortality data of the test was calculated by Trimmed Spearman-Karber (TSK) method (EPA, Version 1.5) to derive the LC50 value and the 95% confidence limit.

During the 96-hour exposure period, there was no death and visible toxic sign observed in the vessels of control group, 3.0 and 4.5 mg/L group. There was lethargy observed in the vessel of 6.7 mg/L, but no death was observed. There were lethargies, discolorations, hemorrhaging, erratic-swimming and lying in the side observed in the vessels of 10 and 15 mg/L. There were only 2 deaths in 10 mg/L, but all test fish in 15 mg/L were dead before the end of the test.

 Under the test conditions, the 96-hour LC50 of test substance to zebra fish was 11.30 mg/L with the 95% confidence limit of 10.20-12.51 mg/L and based on analytically confirmed nominal concentrations.