Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

May 26th to June 29th, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Reliability of original study is 1

Justification for type of information:

Justification for Read Across is given in Section 13 of IUCLID

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of test media including solvent control group and control group were taken from alternating test replicates on days -1 and during the exposure on study days 0, 9, 15, 22 and 28. Additional samples were taken on study 1 from the nominal test item concentration 0.301 mg/L as the recovery on day 0 was in the higher area of the nominal range. The stock solution was sampled and analyzed from freshly prepared and corresponding 7 days aged stock solution of one application interval.
Sampling and pre-treatment: at each sampling day 2 samples were taken per (alternating) test replicate. For each sample 50 ml of each test item concentration, the solvent control and the control were sampled. For analysis of fresh and aged stock solutions, approx. 2 ml of each stock solution were sampled.

Details on test solutions:

An equilibration period of 15 days was carried out prior to start of the exposure and monitored analytically at 7 time points. As the recoveries were not in the expected range in the first measurements, equilibration was continued until most measured concentrations were within the range of ±20 % of the nominal concentration (Day -1). Test solutions flowed through the test vessels for 13 days prior to the initiation of the exposure. Actual test concentrations were determined on study days -1 of the study.
Solvent: Dimethyl sulfoxide (DMSO) was used as a solvent. The solvent concentration was the same in each test concentration and the solvent control (0.10 ml/l).
Stock solutions: A stock solution of 100 g/l was prepared in DMSO. An appropriate amount of the test item was weighed out and transfer red with an appropriate amount of the solvent into a glass flask. The solution was treated with ultrasound for approx. 90 minutes at 26 °C (the test temperature) whilst eventually agitating until it was visually clear. Further stock solutions were prepared by dilution with DMSO. The stock solutions were prepared in appropriate intervals of 7 days. Syringes were filled with the freshly prepared stock solutions or pure DMSO for the solvent control every 7 days.

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 reared at the laboratory from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany
- Maintenance of brood fish: A breeding stock of unexposed, mature zebrafish with an age of approx. 11 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration >60 % of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle (2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum; Daphnia magna, juvenile and adult daphnids, ad libitum; dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.

- Water: Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters:
Total hardness: 10 – 250 mg CaCO3/l
pH-value: 6.0 – 8.5
Alkalinity: 0.6 mmol/l (recent measurement: 2020-01-21)
Acidity: 0.2 mmol/l (recent measurement: 2020-01-21)
Conductivity: 149 μS/cm (recent measurement: 2020-01-21)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Spawning: 15–35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate <5 % during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30 %). About 800 eggs were taken and washed in dilution water. Eggs originated from at least 3 different spawnings
- Fertilization check: Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded.
- Introduction of eggs: Only fertilized eggs with more than 2 cells (4, 8, 16 and 32 cell blastomers) were placed directly in the test vessels. 20 eggs were introduced by random per replicate (corresponding to 80 eggs per treatment group). Fertilized eggs were less than 24 h old at experimental starting. The distribution of eggs to the concentration groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs.
- Start of exposure: The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs, resulting in a total of >120 eggs per test concentration).

POST-HATCH FEEDING
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34). Feeding started 3 days after the beginning of hatch (on study day 5 (post-hatch day 1)). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 8 times daily). 1 day after start of feeding brine shrimp nauplii (48 h old) were additionally fed until the end of the test (2 – 8 times daily).
Brine shrimp nauplii origin, breeding conditions: Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D-67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/l, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water. Feeding ad libitum was carried out.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

34 days (30 days post hatch), depending on post-hatch day 0 (study day 4).

Hardness:

60 to 100 mg CaCO3/l

Test temperature:

25.5-26.8 °C

pH:

7.48-7.90

Dissolved oxygen:

75-95 %

Nominal and measured concentrations:

0.301 - 0.723 - 1.74 - 4.17 - 10.0 mg/l, corresponding to time-weighted arithmetic mean measured concentrations of 0.339, 0.796, 1.72, 4.41 and 10.8 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
glass aquaria of 8.7 l provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 l

- Cleaning: the test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and biodegradation of the test item. Furthermore, the mesh coated fittings were cleaned once per day. Cleaning started on study day 1.
- Aeration:
the dilution water supply tank was aerated. No additional aeration of the test vessels was provided.
- Type of flow-through: Membrane piston pumps provided the water flow. Precision syringe pumps were used for the introduction of stock solutions of the test item dissolved in DMSO into the mixing chamber. The stock solutions and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L) where the eggs/fish were exposed. One mixing chamber was used for one test aquarium (replicate).
- Renewal rate of test solution (frequency/flow rate):
the accuracy of the water flow was checked prior to start of the exposure and three times per week thereafter. Water exchange in the test aquaria was about 10 test vessel volumes per day (3.125 l/h).
- No. of fertilized eggs/embryos per vessel:
20
- No. of vessels per concentration (replicates):
four
- No. of vessels per control (replicates): four
. Dilution water (without test item)
- No. of vessels per solvent control (replicates):
four. A solvent control with 0.10 ml DMSO/l dilution water was prepared and tested under the same conditions as the test groups
- Biomass loading rate:
20 eggs per replicate. A loading rate not exceeding 0.5 g/l wet weight fish per 24 hours and not exceeding 5 g/l of solution at any time was maintained

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine
- Total organic carbon:
mean TOC was 1.42 mg/l
- Intervals of water quality measurement:

Once per hour: temperature in the dilution water, measured in one control vessel
At least 3 times per week: Determination of dissolved oxygen in all replicates of each test group and check of flow rates of the test media (variation <10 % throughout exposure
Weekly: pH-value and temperature in all replicates of each test group, TOC and Chlorine from dilution water. Total hardness in one replicate of control, solvent control and the highest test item concentration.
The light intensity on the surface of the test aquaria was measured at the start of the exposure

OTHER TEST CONDITIONS
- Photoperiod:
a daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
- Light intensity:
300 ± 150 Lux

EFFECT PARAMETERS MEASURED
All biological parameters were observed daily. Dead larvae/fish and coagulated or dead eggs were removed daily, if observed
- Hatching: the number of hatched larvae was determined daily until study day 7. All embryos hatched were counted as hatched, even if they had died directly afterwards. Eggs were only removed, when mortality of eggs/embryos was observed. On study day 4, 95 % of the control and 98 % of the solvent control larvae had hatched. Therefore, study day 4 was defined as posthatch day 0 (= PHD 0). For evaluation of hatch, all hatched larvae (even dead ones) were counted. The cumulative number of hatched larvae was used for evaluation.
- Mortality: Criteria for mortality vary according to life stage: For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded. For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded
- Further effects: abnormal appearance and behavior were also recorded daily. The number of larvae or fish showing abnormality of body form was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. quiescence, hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate.
- Measurement of fish size: at the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was measured to the nearest 0.5 mm with graph paper. The total length (from the tip of the snout to the tip of the longer lobe of the caudal fin) was measured.
- Measurement of fish wet weight: at the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis to the nearest 0.1 mg. Fish were blotted on paper towels to remove excess moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.

RANGE-FINDING STUDY
- Test concentrations: A non-GLP preliminary range finding test was conducted at the test facility as a shortened earlylife stage test under flow-through conditions over a period of 16 days. An equilibration period over 6 days was carried out prior to the start of the exposure. Exposure was started by placing fertilized eggs into the test replicates. A solvent control and two test concentrations of the test item of 1 and 10 mg/l were tested. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control. The test item concentrations were analytically verified via HPLC-DAD on days -3, 6 and 13.

POST-HATCH DETAILS
- Begin of post-hatch period:
study day 4
- No. of hatched eggs (alevins)/treatment released to the test chamber:
For the whole study (including the range finding tests) 722 healthy eggs/fish were used

FERTILIZATION SUCCESS STUDY
- Number of eggs used:
20 for each replicate

Reference substance (positive control):

no

Remarks:

No reference item is recommended for this test according to the guideline.

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

10.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

hatch

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

4.41 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

4.41 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

10.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival and overall survival

Details on results:

- Egg Fertilization Rate: the egg fertilization rate determined on study day 0 (start of the exposure) was 93 %. Eggs were fully covered with the respective test solutions during fertilization check.

- Hatch and Definition of Post Hatch Day 0: hatch began on study day 2 in all test treatments but the solvent control. Hatch continued until study day 5. The hatch of the control, solvent control and all test item concentrations was completed until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95 % in the control and 98 % in the solvent control.

- Swim-up: Swim-up was observed for a 3-day period from study days 4 to 6. Newly hatched fry began to swim up on study day 4 (PHD 0). On study day 6 (PHD 2), all surviving larvae had
swum up. No statistical analysis of swim-up data was carried out.

- Fry Survival (Post-Hatch Survival): the post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥75 %). The fry survival (post-hatch survival) at the end of the study was 93 % in the control and 95 % in the solvent control. No concentration-related decrease of the post-hatch survival was detected with increasing test concentrations.

- Overall Survival: The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 11 % in the control group and 6 % in the solvent control group, thus fully meeting the validity criteria of the guideline given in section 9. No concentration-related decrease of the overall survival was detected in the test item concentrations.

- Fry Growth: Fry growth, expressed as length and wet weight measurements, was measured on study day 34 (PHD 30) from all survivors

- Morphological and Behavioral Effects: No biologically significant morphological and behavioral effects were observed in the test item concentrations 1.72 and 4.41 mg/l. In the control, the solvent control and the test item concentration 0.339 mg/l, isolated events of side position (S) and coordination issues (C) were observed from study day 9 up to study day 27. In the concentrations 0.796 and 10.8 mg/l, isolated events of coordination issues (C) were observed from study day 11 up to study day 18.

- Biomass Loading: The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure. The maximum biomass at the end of the exposure was determined in replicate 1 of the test concentration 0.796 mg/l: 1177.2 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 157 mg/l. The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 15.7 mg per liter and day. These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.

- Measured Concentrations in the Test Vessels during Exposure: the measured concentrations of the test item were in the range of 87 to 124 % of the nominal concentrations in all samples. In samples of the test item concentration 0.301 mg/L, values outside the range of ±20 % of the nominal concentration were measured. As quality control samples showed a good procedural recovery for the analytical method and sample preparation, the variation of the measured values of the low concentration are presumably related on inherent effects of the test item in the flow through system, such as possible sorption or biological residues in the water. The time-weighted arithmetic mean measured concentrations were calculated for both test concentrations and used for calculation of the effect values.

- Measured concentrations of the freshly prepared stock solution was 90 % of the nominal value. Measured concentrations of the old stock solutions (4 days and 7 days old stock solutions) were in the range of 102 to 107 % of the nominal values.

- Metabolite Screening: An aged sample of the highest test concentration was measured via LC-Q-TOF (non-GLP method) for confirmation of two isomers of the test item resulting in two chromatogram peaks at retention time approx. 5.6 (1012.2075 m/z) and 5.9 min (1012.2081 m/z) in both freshly prepared and aged samples. It is noteworthy that on basis of the measured accurate masses, only the molecular formula was predicted. The chemical structure of the compounds has not been determined. Besides these two isomers, no significant metabolites have been identified in aged samples. In comparison to control samples, an additional compound inherent in the test item was found to be present in freshly prepared and aged samples (retention time approx. 8.2 min (394.1786 m/z), presumably resulting from an impurity.

Reported statistics and error estimates:

Hatch and Definition of Post Hatch Day 0: applied for the total number of test organisms that have hatched on study days 2,3,4,5. Chi2 2x2 Table Test with Bonferroni Correction (α=0.05) was done for statistical analysis of hatch after 2, 3, 4 and 5 days. No statistically significant differenceswere found between the controls and the test concentrations. NOEC and LOEC were determined to be 10.8 mg/l and >10.8 mg/l (time-weighted arithmetic mean measured), respectively
Fry survival: Chi2 2x2 Table Test with Bonferroni Correction (α=0.05) was performed for statistical analysis of data on study day 34 (PHD 30). No statistically significant differences were found between the control groups and the test item concentrations. NOEC and LOEC were 10.8 mg/l and >10.8 mg/l(time-weighted arithmetic mean measured), respectively. The LC50-value for post-hatch survival on study day 34 (PHD30) was >10.8 mg/l.
Overall survival: Chi2 2x2 Table Test with Bonferroni Correction (α=0.05) was performed for statistical analysis of data on study day 34 (PHD 30). No statistically significant differences were found between the control groups and the test item concentrations. NOEC and LOEC were 10.8 mg/l and >10.8 mg/ (time-weighted arithmetic mean measured), respectively. The LC50-value for overall survival on study day 34 (PHD 30) was >10.8 mg/l.
Fry growth: statistical procedure applied for length data (α=0.05) the time-weighted arithmetic mean measured concentrations 1.72 to 10.8 mg/l showed significant differences for length. However, as the inhibition was below 10 % in the concentration 1.72 mg/l and 4.41 mg/l, respectively, it was considered not biologically relevant. The Williams multiple sequential t-test procedure showed a statistically significant difference in the time-weighted arithmetic mean measured concentration 10.8 mg/l for fresh weight. NOEC and LOEC (time-weighted arithmetic mean measured) for wet weight and length was determined to be 4.41 mg/l and 10.8 mg/l, respectively.

PHYSICOCHEMICAL DATA

- Dissolved Oxygen: the dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 88 - 90 % and ranged from 75 to 95 % during the exposure period.

- Water Temperature: during the exposure the water temperature was recorded continuously (once per hour) with a data logger. The mean temperature was 26.3 °C. The minimum temperature was 25.5 °C and the maximum temperature was 26.8 °C. The water temperature did not differ by more than ± 1.5 °C between successive days during the test. The validity criterion for the parameter temperature was fulfilled. The mean water temperature measured once per week from all replicates during the exposure period was 26.0 °C for the control. The minimum and maximum measured temperature for the control were 25.5 and 26.5 °C, respectively.

- pH values: the mean pH-values in the control, solvent control and test item groups were between 7.60 and 7.73 and ranged from 7.48 to 7.90 during the exposure period.

- Total Hardness: total hardness of the test media was measured from the control, solvent control and the highest nominal test concentration of 10.0 mg/l. Total hardness was measured once per week. The mean total hardness was 67 mg CaCO₃/l and ranged from 60 to 100 mg CaCO₃/l in the control, solvent control and the highest test concentration.

- Residual Chlorine: it was measured from the dilution water supply tank on study days 2, 8, 15, 23 and 30 was < 0.01 mg/l.

- Total Organic Carbon (TOC) of the Dilution Water: the total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 2, 9, 16, 22 and 30. The mean TOC was 1.42 mg/l throughout exposure.

- Flow Rates: the mean flow rate through the mixing chambers of all test item and control groups was 3.12 ± 0.106 l/h and the individual values ranged from 2.82 to 3.60 l/h.

A precision syringe pump was used for introduction of the stock solution to the mixing chambers. At any renewal of the syringes the proper function of the pump and the applied volume was checked by the syringe volume indicator.

- Light intensity: light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 181 to 421 lux (mean: 284 lux).

Validity criteria fulfilled:

yes

Remarks:

DO saturation was >60 %;Water temperature: did not differ by more than 1.5 °C; Hatching success >70 %; Post-hatch survival ≥75 %; effect levels are expressed based on the arithmetic mean measured test item concentrations;

Conclusions:

NOEC (hatching success) = 10.8 mg/l (time-weighted arithmetic mean measured)
NOEC (fry length, fry weight) = 4.41 mg/l (time-weighted arithmetic mean measured)
NOEC (post hatch survival) = 10.8 mg/l (time-weighted arithmetic mean measured)
NOEC (overall survival) = 10.8 mg/l (time-weighted arithmetic mean measured)

Executive summary:

The effects of the test item on the early-life stage of fish (Danio rerio/Zebrafish) were determined at the test facility according to OECD Guideline 210. Dimethylsulfoxid (DMSO) was used as solvent with a concentration of 0.10 ml/l dilution water. Stock solutions in DMSO with nominal concentrations of 100, 41.7, 17.4, 7.23 and 3.01 g/l were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.301, 0.723, 1.74, 4.17 and 10.0 mg/l. The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits. On study day 4, 95 % of the control and 98 % the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0) . Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival. Specific analysis of various concentrations of the test item in the test media and the controls was carried out via HPLC-DAD. The test media were sampled and analyzed prior to exposure on days -1 and during the exposure on study days 0, 1, 9, 15, 22 and 28. The measured concentrations of replicates and test media on study days 0 to 34 (last sampling on study day 28) were in the range of 87 to 124 % of the nominal concentrations. Since the measured concentrations were partly out of the range of ±20 % of the nominal concentration, the time-weighted arithmetic mean measured concentrations of the test item were calculated, resulting in 0.339, 0.796, 1.72, 4.41 and 10.8 mg/l, respectively. The DMSO stock solutions were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solution. All effect values are given based on the time-weighted arithmetic mean of the test item. The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences. No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis. The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results.

The test item partly caused significant effects on Zebrafish in an early life stage test. For the parameters hatch, post-hatch survival and overall survival the NOEC was 10.8 mg/L. Therefore, the respective LOECs were determined to be > 10.8 mg/l. For the parameter fry growth (expressed as length and fresh weight) the NOEC was 4.41 mg/l. Therefore, the LOEC for this parameter was determined to be 10.8 mg/l.

Description of key information

NOEC (fry length, fry weight) = 4.41 mg/l (TWA, mean measured)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.41 mg/L

Additional information

No data are available for the evaluation of the long-term toxicity to fish of the substance. For this reason, data on Similar Substance OB 1 -MSA is used. Read across justification is given in Section 13 of IUCLID.

 

The effects of the test item on the early-life stage of fish (Danio rerio/Zebrafish) were determined according to OECD Guideline 210. Dimethylsulfoxid (DMSO) was used as solvent with a concentration of 0.10 ml/l dilution water. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.301, 0.723, 1.74, 4.17 and 10.0 mg/l. The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits. On study day 4, 95 % of the control and 98 % the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0). Hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival were assessed. Specific analysis of various concentrations of the test item in the test media and the controls was carried out via HPLC-DAD. The measured concentrations of replicates and test media on study days 0 to 34 (last sampling on study day 28) were in the range of 87 to 124 % of the nominal concentrations. Since the measured concentrations were partly out of the range of ±20 % of the nominal concentration, the time-weighted arithmetic mean measured concentrations of the test item were calculated, resulting in 0.339, 0.796, 1.72, 4.41 and 10.8 mg/l, respectively.

The test item partly caused significant effects on Zebrafish in an early life stage test. For the parameters hatch, post-hatch survival and overall survival the NOEC was 10.8 mg/L. Therefore, the respective LOECs were determined to be >10.8 mg/l. For the parameter fry growth (expressed as length and fresh weight) the NOEC was 4.41 mg/l. Therefore, the LOEC for this parameter was determined to be 10.8 mg/l.