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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.2019 - 08.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test.

Qualifier:

according to guideline

Guideline:

other: Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

All sorts of analytical grade salt were dissolved in deionised water to obtain the stock solution and standard dilution water as following:
Solution Component Concentration (g/L)
Stock a CaCl2·2H2O 11.76
Stock b MgSO4·7H2O 4.93
Stock c KCl 0.23
Stock d NaHCO3 2.59
standard dilution water The conductivity of the deionised water should not exceed 10μs/cm. 25mL each of solutions (a) to (d) are mixed and the total volume made up to 1 litre with deionised water. The water will be aerated continuously over 24h.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

The test was performed with Gobiocypris rarus which were bred in Bioassay and Safety Assessment Lab, Shanghai Academy of Public Measurement (ID: GR-SAPM-190305). This freshwater fish species is suitable for fish toxicity testing because of their ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species. This species complies with “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test”, “OECD, Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test, 1992” and “GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test”.
For evaluation of the fish quality and experimental conditions, potassium dichromate was tested as a positive control to demonstrate satisfactory test conditions once a year (recently performed on 2019.6.3-6.7) (96h). The 96h LC50 value of the test substance to Gobiocypris rarus (ID: GR-SAPM-190305) was 192mg/L (95% confidence limits: 154-239mg/L), which was within the ±2SD range of the QC chart in our lab (82.0-316mg/L).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

21 - 25 °C

pH:

6 - 8.5

Dissolved oxygen:

Dissolved oxygen concentration: > 80% of air saturation value (ASV);

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

Test Conditions

Water
Standard dilution water was used. The water was aerated continuously over 24h.

Test vessels
Five litre glass tanks were used containing 4L test solution.

Conditions of exposure
Duration: 96h;
Test method: semi-static and the frequency of renewal was every 24h;
Loading of the test fish: about 0.6g fish/litre;
Temperature: 21.8-22.0℃;
Photoperiod: Light/Dark=12 hours/12 hours;
Dissolved oxygen concentration: 72.5%-92.4%ASV;
Feeding: none.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The actual test substance concentration of test solutions was determined by using a TOC analysis method. The limit of detection (LOD) for the test substance using this method was calculated to be 0.243mg/L, the routine limit of quantification (LOQ) for the test substance using this method was calculated to be 0.902mg/L. It was found that the average recoveries were 104% and 99.9% of the spiked 2.00mg/L and 40.0mg/L samples (TOC concentration) with RSD values of 0.83% and 0.14%, respectively.
In the limit test, the measured concentrations of TOC were below LOQ，LOQ=0.902mg/L. There were no mortalities and abnormalities observed in the fish of the control group and the treatment group during the test.
During the test period, the dissolved oxygen concentrations in the test solutions ranged from 72.5%ASV to 92.4%ASV. The temperature in the test solutions ranged from 21.8℃ to 22.0℃. The pH value in the test solutions ranged from 7.1 to 7.2.

Results with reference substance (positive control):

Reference substance information
Name: Potassium dichromate (Sinopharm Chemical Reagent Co.,Ltd)；
Code: REF002-2018-01;
CAS No.: 7778-50-9;
Molecular formula: K2Cr2O7;

96h LC50 and its 95% confidence limits of the reference substance to Gobiocypris rarus
Duration (h) LC50 (mg/L) 95% confidence limits (mg/L)
Lower Upper
96 192 154 239

Validity criteria fulfilled:

yes

Conclusions:

There was no acute toxicity of the saturated solution in the limit test. The test results showed that the 96h LC50 for the test substance LICOCARE RBW 300 FL TP to Gobiocypris rarus was higher than the concentration of the saturated solution (the measured concentration of TOC were below LOQ, LOQ=0.902mg/L) under the tested conditions

Executive summary:

According to “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test”, “OECD, Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test, 1992” and “GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test”, the acute toxicity of the test substance LICOCARE RBW 300 FL TP (KLEC20181026-2692) to Gobiocypris rarus was determined by a semi-static test.

A nominal concentration of 100mg/L of the test substance was prepared and stirred (via magnetic stirrer) in the dark for 72h (the vortex depth was 1/3 of that of the mixture column) followed by filtration using a 0.45μm nitrocellulose membrane to make a saturated solution of the test substance in the test water (referred to as “saturated solution” in this report). Based on the results of the range-finding test, the saturated solution was used to perform the limit test with a blank control. The test duration was 96h and the frequency of renewal was every 24h.

There were no mortalities and abnormalities observed in treatment group during the test period. No mortality occurred in the control during the test and the dissolved oxygen concentration was greater than 60.0% of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, The measured concentrations of total organic carbon (TOC) analyzed in the test solutions were below the limit quantitative(LOQ，LOQ=0.902mg/L). There was no acute toxicity of the saturated solution in the limit test. The 96h LC₅₀ of the test substance to Gobiocypris rarus was higher than the concentration of the saturated solution (the measured concentration of TOC were below LOQ，LOQ=0.902mg/L) under the tested conditions.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2020-07-19 to 2020-07-24 with the definitive exposure phase from 2020-07-20 to 2020-07-24 at the test facility

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item Licocare RBW 300 FL VITA

Batch number DEF2114527

Purity (certified) 99.75% (w/w) in aqueous solution

Density ca. 0.99 mg/cm3 (23 °C)

pH 6.53 (23 °C)

Appearance yellow solid flakes

Water Solubility < 1 g/L

Stability under test
conditions Not specified

Expiry date 2020-11-05

Recommended storage Store container tightly closed in a cool, well-ventilated place

Analytical monitoring:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item.

Vehicle:

no

Details on test solutions:

Limit loading
A water accommodated fraction (WAF) of 100 mg/L was tested as a threshold concentration in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EL50 (0-48 h) = >100 mg/L) and of an alga toxicity test (ErL50 (0-72 h) = > 100 mg/L).

Water Accommodated Fraction
A water accommodated fraction (WAF) was prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018). Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

 
Preparation of the WAF
Two WAFs were prepared with nominal loadings of 100 mg/L. An appropriate amount of the test item was weighed out and transferred into glass flasks with an appropriate amount of dechlorinated water. The test item dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and a separation phase of at least 1 hour of standing at room temperature, the WAF was removed by siphoning from the approximate center of the water bodies and pooled.
After siphoning, the WAF was checked via laser beam (Tyndall effect) for undissolved test item. Presence of undissolved test item was observed. Therefore, undissolved particles were removed by filtration (folded filter MN615¼, MACHEREY-NAGEL). The following WSF was used in the test. The Tyndall effect was positive.
The preparation of the WSF was carried out beginning one day prior to the start of the exposure (day -1) and one day prior to each renewal of the test solutions.

Control
7 fish in dilution water (without test item but treated in the same way as the test solution) were tested under the same test conditions as the test replicate.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 1.80 cm
- Weight at study end (mean and range, SD): Average body weight: 0.063 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test. No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test.

- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 67 mg CaCO3/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

No analytical monitoring was carried out due to the low solubility of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume:
Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates.
Test volume: About 2.0 L per vessel

- Aeration: No aeration was provided
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.221 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerate for at least 24 h to remove chlorine.

Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.2 mmol/L (recent measurement 2020-07-16)
Alkalinity: 0.5 mmol/L (recent measurement 2020-07-16)
Conductivity: 157 µS/cm (recent measurement 2020-07-16)


- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were
measured in all test vessels at the start and the end of exposure as well as daily and after renewal of the test media from fresh and old test media.
Chlorine and nitrate were measured from each batch of dilution water used in the test.
Total hardness and TOC were determined at the start of the exposure in the dilution water. During the test, the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure. The solution appearance and behavior was observed daily before and after renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 540 - 1000 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2 ± 0.5 h, 5 ± 1 h and 24 ± 2 h after the start of the exposure. On days 2-4 of the test, all vessels with living fish were inspected twice per day.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: See tables below
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solution was visibly clear throughout the period of exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation The LL-values after 96 h were empirically derived from the raw data of the limit loading.

Software All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:

The following software was used for calculations:
Excel, MICROSOFT CORPORATION

Sublethal observations / clinical signs:

 Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels

                   (n = 7, number of fish)

 

Nominal Test item loading [mg/L]	Effect *	Number of fish affected at observation time [hours]
		day 0		day 1		day 2		day 3		day 4
		2	4	22	25	48	50	70	74	93	96
100	(1)	7	7	7	7	7	7	7	7	7	7
Control	(1)	7	7	7	7	7	7	7	7	7	7

               *)  The numbers in brackets correspond to the following observations: 

              (1)       = Normal behavior

 

Cumulative Mortality [%] in the Test Vessels

Nominal Test item loading [mg/L]	Cumulative mortality [%] at observation time [hours]
	day 0		day 1		day 2		day 3		day 4
	2	4	22	25	48	50	70	74	93	96
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The effect level is given based on the nominal test item loading of Licocare RBW 300 FL VITA. The threshold concentration of the test item at the nominal loading 100 mg/L was found to have no lethal or toxic effects on zebrafish.
In conclusion, the LL0 corresponds to the nominal loading of ≥ 100 mg test item/L, the LL50 was > 100 mg/L.

Executive summary:

The acute toxicity of the test item Licocare RBW 300 FL VITA to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2020-07-19 to 2020-07-24 with the definitive exposure phase from 2020-07-20 to 2020-07-24 at the test facility.

 

The test item consists of yellow solid flakes. A limit concentration of 100 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EL₅₀ (0-48 h) = >100 mg/L) and of an alga toxicity test (E_rL₅₀ (0-72 h) = > 100 mg/L).

 

The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours. Seven test organisms were exposed to the test loading and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test solutions were visually clear throughout the study.

 

All validity criteria of the test guideline were met. The results of the study are summarized in the table below. The test item does not show toxic effects on fish at the test loading 100 mg/L.

LL-Values (96 hours) of the Test Item

                    Based on the nominal loading of the test item [mg/L]

 

LL0 = Highest test item loading with  0% mortality after 96 h	≥ 100 mg/L
LL50 = Highest test item loading with  50% mortality after 96 h	> 100 mg/L

 

Description of key information

 

Study 1

The acute toxicity of the test item Licocare RBW 300 FL VITA to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2020-07-19 to 2020-07-24 with the definitive exposure phase from 2020-07-20 to 2020-07-24 at the test facility.

 

The test item consists of yellow solid flakes. A limit concentration of 100 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EL₅₀ (0-48 h) = >100 mg/L) and of an alga toxicity test (E_rL₅₀ (0-72 h) = > 100 mg/L).

 

The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours. Seven test organisms were exposed to the test loading and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test solutions were visually clear throughout the study.

 

All validity criteria of the test guideline were met. The results of the study are summarized in the table below. The test item does not show toxic effects on fish at the test loading 100 mg/L.

 

LL-Values (96 hours) of the Test Item

                    Based on the nominal loading of the test item [mg/L]

 

LL0 = Highest test item loading with  0% mortality after 96 h	≥ 100 mg/L
LL50 = Highest test item loading with  50% mortality after 96 h	> 100 mg/L

 

 

Study 2

According to “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals–Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test”, “OECD, Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test, 1992” and “GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test”, the acute toxicity of the test substance LICOCARE RBW 300 FL TP (KLEC20181026-2692) to Gobiocypris rarus was determined by a semi-static test.

A nominal concentration of 100mg/L of the test substance was prepared and stirred (via magnetic stirrer) in the dark for 72h (the vortex depth was 1/3 of that of the mixture column) followed by filtration using a 0.45μm nitrocellulose membrane to make a saturated solution of the test substance in the test water (referred to as “saturated solution” in this report). Based on the results of the range-finding test, the saturated solution was used to perform the limit test with a blank control. The test duration was 96h and the frequency of renewal was every 24h.

There were no mortalities and abnormalities observed in treatment group during the test period. No mortality occurred in the control during the test and the dissolved oxygen concentration was greater than 60.0% of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, The measured concentrations of total organic carbon (TOC) analyzed in the test solutions were below the limit quantitative(LOQ，LOQ=0.902mg/L). There was no acute toxicity of the saturated solution in the limit test. The 96h LC₅₀ of the test substance to Gobiocypris rarus was higher than the concentration of the saturated solution (the measured concentration of TOC were below LOQ，LOQ=0.902mg/L) under the tested conditions.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L