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Ecotoxicological information

Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-07-29 to 2021-10-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
July 26, 2013.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Test item name: Butyl methoxydibenzoyl methane
- Chemical name: 1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3-dione
- CAS Number: 70356-09-1
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Triplicate samples were taken from the test medium out of each of the four replicates and duplicate samples were taken directly from the outlet pipe of the mixing vessel of the dosing unit. Samples were taken at day -8, -4 and -2. Additionally, single samples were taken from the freshly prepared application solution. During the exposure period, triplicate samples were taken out of all replicates of the test medium at day 0 (start of the exposure period) and on 15 further sampling dates, i.e. at least every three to four days. At the same sampling dates, duplicate samples were taken out of all replicates from the control and solvent control. Additionally, at all application solution renewal periods, single samples were taken from the freshly prepared and aged application solutions.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples the test item proved to be stable under these storage conditions.
Vehicle:
yes
Remarks:
N,N-dimethylformamide p.a. (DMF, final concentration of the solvent in the test medium was 50 μL/L)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: computer controlled automated flowthrough dosing system (Pequitec MSR System, 4852 Rothrist, Switzerland); three individual dosing units for test medium/water supply for single test concentration, negative control and solvent control
- Dosing: concentrated application solution of the test item in a mixture of DMF and purified water (1:1) application solutions, renewed 6 times during the test period; medium prepared batch-wise by mixing defined volumes of application solution in test water, batch of test medium then distributed to four replicate test vessels
- Cycle of test medium preparation: A cycle for test medium preparation in the dosing unit started by dosing 100 µL application solution by use of a syringe pump into a mono-block glass mixing chamber filled with 1000 mL of test water. In the mixing chamber, the test medium was intensively stirred by a magnetic stirrer for 3 minutes to ensure that the application solution was homogeneously distributed in the test medium. As the application solution was a 1:1 mixture of DMF and purified water, the final DMF concentration in the test medium was 50 µL/L. After stirring, the test medium was channelled from the mixing chamber to the 4 replicates by a Teflon tube, which was moved by an automated extension arm from one replicate to the next. By repeated turns of the extension arm, the test medium volume from the mixing chamber was equally distributed to the replicates in several identical aliquots. In this way, the test medium flow was the same for all four replicates. After the complete distribution of the test medium in the mixing chamber, a new test medium preparation cycle started. The frequency of test medium preparation cycles was 5 minutes, i.e. 12 cycles per hour (= 288 cycles per day). Per cycle, 1 litre medium was prepared in the mixing vessel. Thus, in total 288 litre test medium per day were prepared for each treatment. At the beginning of the test, when 400 mL glass vessels were used, only 300 mL test medium of the 1000 mL batch were used and 700 mL were discarded to avoid an excessive water renewal rate, which might stress the larvae and fish. This water flow resulted in a 54-fold water exchange rate per day in each of the four replicate test vessels. When 2-L and 7-L glass vessels were used, the total volume of 1000 mL was distributed to the 4 replicates. This water flow resulted in a 36-fold (for 2-L vessels) and 10-fold (for 7-L vessels) water exchange rate per day in each of the four replicate test vessels. Test water without any addition of test item or solvent flowed at the same rate into the replicates of the control. For the solvent control, DMF at a concentration of 50 µL/L was dosed into test water. Thus, the final DMF concentration in the solvent control and the test medium was the same. Water flow and dosing systems were calibrated before the start of the dosage. The dosing system was visually inspected at least once a day. The correct function was evaluated by volumetric measurements (filling level of the test water in the mixing chamber) and gravimetric measurements (amount of application solution dosed into the mixing chamber).
- Controls: negative control (test medium only), solvent control
- Chemical name of vehicle: N,N-dimethylformamide (DMF)
- Concentration of vehicle in test: 50 µL/L
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the medium in the mixing chamber of the dosing unit and in the test vessels. The test medium appeared to be a clear solution throughout the test period.
- Dosage pre-experiment: To investigate the technical feasibility of dosing the test item at the limit of its solubility in test water, a dosage pre-experiment was conducted in a flow-through system with three nominal test item concentrations, i.e. at 2.0, 4.0 and 6.0 µg/L. The solubility limit of the test item was assumed to be approximately 2 µg/L and was based on results of a previous study performed with butyl methoxydibenzoyl methane. The flow-through system was the same as was later used in the main test. A mixture of N,N-dimethylformamide (DMF) and purified water in ratio 1:1 was used as solvent for the preparation of a concentrated application solution. Different test vessels with 2 L and 7 L test medium volume were used, i.e. the same size of test vessels that were used later during the course of the main test. The test medium flow through the different vessels corresponded to renewal rates of 23-fold (2 L vessels) and 6-fold (7 L vessels) renewals per day. During a period of 4 days, test item concentrations were measured at Days 2, 3 and 4, reaching recoveries in the range of 70 to 88 %, 49 to 64 % and 68 to 85 % of nominal for the test item concentration of 2.0 µg/L, 4.0 µg/L and 6.0 µg/L, respectively.
- Stirring Experiments: The solubility limit in test water was based on M7-medium, which was used as test water in a Daphnia reproduction test. In order to confirm the solubility limit of the test item in the type of test water as used in the main study further pre-experiments were performed. In a first solubility test, three individual suspensions of the test item in test water with a loading rate of 100 mg/L were mixed by stirring intensively for 3, 24, and 96 hours. After stirring, the suspensions were either centrifuged at 3500 rpm or filtered through a 0.45 µm membrane filter. The filter was saturated with 200 mL test medium. The analytically determined concentrations of dissolved test item in the filtrates were 3.2 µg/L (3 hours), 1.4 µg/L (24 hours) and 4.6 µg/L (96 hours). In the supernatants after centrifugation, test item concentrations of 161 µg/L (3 hours), 198 µg/L (24 hours) and 628 µg/L (96 hours) were found. From this solubility test, it was concluded that centrifugation seems to lead to incomplete separation of the undissolved fraction. Therefore, only the analytical results from the filtrates were considered for determining the test design for further testing. The analytically determined concentration of dissolved test item in the test water was higher after 96 hours of stirring (4.6 µg/L) compared to the value at 24 hours of stirring (1.4 µg/L). Therefore, the plateau of solubility may not have been reached and it was decided to confirm the solubility limit in a further prolonged solubility test. For this purpose, four individual suspensions of the test item in test water with a loading rate of 100 mg/L were mixed by stirring intensively for 4, 7, 10, and 14 days at room temperature in the dark. After stirring, the suspensions were filtered through a 0.45 µm membrane filter. The filter was previously saturated with 500 mL test medium. The analytically determined concentrations of dissolved test item in the filtrates were 1.3 µg/L (4 days), 4.2 µg/L (7 days), 1.4 µg/L (10 days) and 1.4 µg/L (14 days); the higher value after 7 days can be attributed to the concentration of 7.4 µg/L measured in one of the two replicates (concentration in second replicate: 1.1 µg/L), which was considered as an outlier. Thus, a plateau was reached after 4 days of stirring, i.e. no consistently higher concentrations of dissolved test item could be achieved with extended stirring periods. The mean concentration over all stirring periods (i.e. the mean concentration of the plateau) was approximately 2 µg/L. This test item concentration corresponded approximately to the water solubility as reached in the first stirring experiment after 3 hours. An additional suspension with the loading rate of 100 mg/L was mixed by stirring intensively for 4 days and used for testing the filter saturation of the 0.45 µm membrane filters. For this purpose the membrane filter was preconditioned with 500 mL test medium and analytical samples were taken from the filtrate. With the same filter, three consecutive filtration steps were conducted and further samples were taken from each filtrate. The analytically determined test item concentrations were 2.3, 1.7 and 2.3 μg/L after the 1st, 2nd and 3rd filtration step, respectively. These results showed that the test item concentrations that were achieved in the course of three filtration steps remained constant and that the filter was saturated already after one saturation step with a volume of 500 mL test medium. Summarizing the results of the two stirring experiments it was concluded that the maximum concentration of dissolved test item in test water used for the present study is in the range of 1.1 to 4.6 µg/L. The mean concentration of all measurements of the two stirring experiments was calculated to be 2.1 µg/L.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: originally obtained from IME, Fraunhofer Institute, 57392 Schmallenberg, Germany; held at laboratory in accordance with the Swiss Animal Protection Law
- Breeding conditions: held in an aquarium in reconstituted water, 16-hour light to 8-hour dark photoperiod with a 30-minute transition period, fed with commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49324 Melle / Germany) and brine shrimp larvae (Artemia salina)
- Health of brood hatch: regularly visually checked for abnormal behavior, diseases or mortality; no visible abnormalities observed in brood batch during two months before test start, no medication applied

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: glass dishes covered with stainless steel mesh placed on bottom of aquarium one day prior to test start (day -1) for collecting newly fertilized eggs; mesh separated fertilized eggs (which sank to the bottom into the glass dishes) from the parent fish; spawning started in the morning on day 0 (start of the test = introduction of the eggs into the test medium) after turning on the light.
- Subsequent handling of eggs: collected from glass dishes, randomly exposed to different replicates of test medium, control and solvent control; exposure to test medium started within about 2 hours after fertilization

POST-HATCH FEEDING
- Start date: day 5
- Type/source of feed:
Hatching period day 0 to 4: no food for embryos and newly hatched larvae
Starting from day 5: living ciliates (raised at IES Laboratories)
Starting from day 12: additionally Tetra Min dry powder
Starting from day 13: additionally freshly hatched larvae of Artemia salina (raised at IES Laboratories)
Starting from day 17: freshly hatched larvae of Artemia salina and Tetra Min dry powder
- Amount given: ad libitum while minimizing the surplus
- Frequency of feeding: fed at least three times each working day, on weekends at least twice per day; not fed 24 hours prior to the end of the test to allow clearance of the digestive tract before weighing the fish
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
34 d
Remarks on exposure duration:
hatching period from Day 0 to Day 4 plus a 30-day post hatch period
Post exposure observation period:
Post exposure observation not performed
Hardness:
125 mg/L as CaCO3 (hardness of test water)
Test temperature:
26.4 to 26.7 °C
pH:
7.0
Dissolved oxygen:
7.6 mg/L (mean for limit concentration treatment)
Salinity:
not applicable
Conductivity:
approx. 360 μS/cm (conductivity of test water)
Nominal and measured concentrations:
nominal: 6.0 µg/L; measured: 4.0 µg/L (arithm. mean, 66 % of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: With increasing fish size, the volumes of the test vessels were gradually increased as follows
Days 0-13: Glass vessels with 400 mL water volume
Days 13-18: Glass beakers with 2 L water volume
Days 18-34: Mono block glass aquaria with 7 L water volume
- Aeration: The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached
- Type of flow-through: computer controlled automated flowthrough dosing system (Pequitec MSR System, 4852 Rothrist, Switzerland)
- Renewal rate of test solution: 36-fold (for 2-L vessels) and 10-fold (for 7-L vessels) water exchange rate per day in each of the four replicate test vessels
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM PARAMETERS
- Preparation of dilution water: reconstituted test water consisting of analytical grade salts dissolved in purified water
CaCl2 × 2H2O: 147 mg/L
MgSO4 × 7H2O: 61.5 mg/L
NaHCO3: 32.5 mg/L
KCl: 2.9 mg/L
Water Hardness: 125 mg/L as CaCO3
Alkalinity: 0.4 mmol/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark photoperiod with a 30-minute transition period
- Light intensity: 7.8 and 9.8 µmol s^-1 m^-2.

EFFECT PARAMETERS MEASURED:
- Egg development and hatching rate: The embryonic development and hatching of larvae in the control, solvent control and at the test concentration were recorded daily for all replicate test vessels. Eggs showing a change in color (white non-transparent color caused by coagulation and/or precipitating of protein) and embryos showing no body movement were considered to be dead. Dead eggs and embryos were removed from the test vessels. Percent of hatching success was calculated for each replicate by dividing the number of hatched larvae by the number of inserted eggs.
- Development and survival of larvae and juvenile fish: The survival of the larvae and juvenile fish was observed each day. Mortality criterion for larvae and juvenile fish was lack of reaction to mechanical stimulus. The survival rate of the test fish was calculated for the test concentration and the control and solvent control by dividing the number of surviving fish by the number of larvae hatched (number of larvae hatched on day 4 was set to 100%). The larvae and juvenile fish were also observed for visible abnormalities like abnormal appearance (body form) and abnormal behaviour (e.g. uncoordinated swimming, atypical quiescence and atypical behaviour). Eggs, larvae and fish, which were missing at the observation dates, were considered to be dead. Observations were recorded at least three times per week or at the date of the finding.
- Fish length and fish weight: At the end of the test the test fish were sacrificed and the fish length (total length from the mouth to the end of the tail fin) and the body wet weight were determined for each individual fish. The dry weight of the fish (drying for 24 hours at 60 °C) was determined from the sum of all fish per replicate, since the fish were very light after drying and individual weight measurement was not feasible.
- Mortality

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
For determination of the appropriate test concentration for the main test, a 12-day range finding test under flow-through conditions was performed with the nominal test item concentrations of 2.0, 4.0 and 6.0 µg/L. As the application solutions for this range-finding test were prepared in a mixture of DMF and purified water (1:1), a solvent control with the solvent DMF was run in parallel. The final concentration of DMF in the test media and the solvent control was 50 µL/L.
The flow-through system was the same as was used later in the main test. The experimental conditions regarding type of test water, water temperature and light conditions were identical to the conditions described for the main test. 400 mL glass vessels were used for the exposure of the eggs and larvae. The renewal rate of the test medium was 56-fold per day. The range finder was started with 15 newly fertilized eggs for each of the two replicates per treatment. The larvae were fed with living ciliates and freshly hatched larvae of Artemia salina.

During the range finding test, no statistically significant effects on hatching rate and no mortality or visible abnormalities (e.g. signs of chronic toxic effects, behaviour or appearance) were observed at the hatched larvae. During the range finding test, analytical samples were taken from the test media at the start of the test and at days 3, 7, 10 and 12. To examine the influence of organic matter (bacterial growth, surplus feed), at day 10 samples were taken from used vessels and from newly introduced test vessels after renewal. At day 10, additional samples were taken directly from the outlet of the mixing chamber (test medium without organic matter). Further samples were taken from the freshly prepared and aged application solutions (7-day stability control). The concentrations of the test item in the freshly prepared and aged application solutions were in the range of 96 and 111 % of the nominal values. This demonstrates the stability of the test item in the application solutions over a period of 7 days. In the test media, variation of measured concentrations was observed. This was attributed to adsorption of the test item to food, faeces and bacterial growth, which takes place in a biological system. The comparison of the samples taken from new and used test vessels indicate adsorption of the test item to organic matter. This effect cannot be entirely avoided in a test system with living organisms that have to be fed daily. To further improve the dosing of the test item, technical parameters of the dosing system were adapted, i.e. increase of the water exchange rate, where technically feasible, and extension of the stirring time in the mixing chambers to avoid possible inhomogeneity.

POST-HATCH DETAILS
- Begin of post-hatch period: day 5
Reference substance (positive control):
no
Key result
Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
> 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks:
hatching success after 4 days of exposure
Key result
Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
> 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
survival after 34 days of exposure
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks:
hatching success after 4 days of exposure
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
survival after 34 days of exposure
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks:
mean fish body length after 34 days of exposure
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
mean fish wet weight after 34 days of exposure
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
mean fish dry weight after 34 days of exposure
Details on results:
- Mortality/survival at larval and juvenile stages: survival rate was 87.4 %
- Days to hatch or time to release of young: 4
- Numbers hatched per day: for the treatment (6.0 µg test item/L) the mean hatching rate was 98.8 % (for details please refer to respective table in below section “Any other information on results incl. tables”)
- Observations on body length and weight of young and/or exposed parents at one or more time periods: no effects observed (for details please refer to respective table in below section “Any other information on results incl. tables”)
- Other biological observations: no
- Incidents in the course of the test which might have influenced the results: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional® Version 3.3.0. Hatching rate: Fisher’s Exact Binominal test, two-sided, α = 0.05; mortality of fish: Fisher’s Exact Binominal test, one-sided greater, α = 0.05; fish body length: Two-sample t-test Procedure, two-sided, α = 0.05; fish wet weight: Two-sample t-test Procedure, two-sided, α = 0.05; fish dry weight: Two-sample t-test Procedure, two-sided, α = 0.05. The EC10-values for fish body length, wet weight and dry weight could not be calculated due to the lack of toxicity of the test item at the limit concentration of nominal 6.0 μg/L.

Table 1: Results for test samples from main test






































































































































































































































































































































Sampling Day



Nominal Concentration of Test Item
cnom



Repli-cate



Sample ID



Measured Concentration

x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d]



[µg/L]



 


 

[µg/L]


 

[µg/L]



[%]



0



0 (Control)



A



F46



n.d.



2



< LOQ



n.a.



0 (Control)*



A



F52



n.d.



2



< LOQ



n.a.



6.0



A



F63



2.12



2



4.24



71



6.0



A



F64



1.75



2



3.51



58



6.0



B



F66



1.77



2



3.54



59



6.0



B



F67



1.54



2



3.08



51



6.0



C



F69



1.84



2



3.69



61



6.0



C



F70



1.51



2



3.03



50



6.0



D



F72



1.70



2



3.40



57



6.0



D



F73



1.83



2



3.66



61


 

 



 



 



Mean



3.52



59



3



0 (Control)



B



F76



n.d.



2



< LOQ



n.a.



0 (Control)*



B



F84



n.d.



2



< LOQ



n.a.



6.0



A



F91



1.99



2



3.99



66



6.0



A



F92



1.92



2



3.85



64



6.0



B



F94



1.79



2



3.59



60



6.0



B



F95



1.63



2



3.26



54



6.0



C



F97



2.02



2



4.03



67



6.0



C



F98



2.05



2



4.10



68



6.0



D



F100



1.80



2



3.60



60



6.0



D



F101



2.04



2



4.07



68


 

 



 



 



Mean



3.81



64



5



0 (Control)



C



F108



n.d.



2



< LOQ



n.a.



0 (Control)*



C



F116



n.d.



2



< LOQ



n.a.



6.0



A



F121



1.56



2



3.12



52



6.0



A



F122



2.12



2



4.24



71



6.0



B



F124



2.03



2



4.05



68



6.0



B



F125



2.15



2



4.29



72



6.0



C



F127



1.81



2



3.62



60



6.0



C



F128



2.07



2



4.13



69



6.0



D



F130



1.88



2



3.76



63



6.0



D



F131



1.75



2



3.50



58


 

 



 



 



Mean



3.84



64



*           solvent control


LOQ:    1.22 µg test item /L


n.d.      = not detected


n.a.      = not applicable


The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.


 


 


 


Continued






































































































































































































































































































































Sampling Day



Nominal Concentration of Test Item
cnom



Repli-cate



Sample ID



Measured Concentration

x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d]



[µg/L]



 


 

[µg/L]


 

[µg/L]



[%]



7



0 (Control)



D



F138



n.d.



2



< LOQ



n.a.



0 (Control)*



D



F146



n.d.



2



< LOQ



n.a.



6.0



A



F149



2.04



2



4.07



68



6.0



A



F150



2.11



2



4.22



70



6.0



B



F152



1.96



2



3.92



65



6.0



B



F153



2.16



2



4.32



72



6.0



C



F155



2.19



2



4.37



73



6.0



C



F156



2.48



2



4.97



83



6.0



D



F158



2.02



2



4.04



67



6.0



D



F159



1.96



2



3.91



65


 

 



 



 



Mean



4.23



70



10



0 (Control)



A



F160



n.d.



2



< LOQ



n.a.



0 (Control)*



A



F168



n.d.



2



< LOQ



n.a.



6.0



A



F177



1.83



2



3.66



61



6.0



A



F178



2.01



2



4.02



67



6.0



B



F180



2.01



2



4.02



67



6.0



B



F181



2.02



2



4.04



67



6.0



C



F183



2.13



2



4.26



71



6.0



C



F184



2.00



2



3.99



67



6.0



D



F186



1.98



2



3.96



66



6.0



D



F187



2.09



2



4.19



70


 

 



 



 



Mean



4.02



67



12



0 (Control)



B



F190



n.d.



2



< LOQ



n.a.



0 (Control)*



B



F198



n.d.



2



< LOQ



n.a.



6.0



A



F205



2.52



2



5.03



84



6.0



A



F206



2.24



2



4.48



75



6.0



B



F208



2.59



2



5.17



86



6.0



B



F209



2.32



2



4.64



77



6.0



C



F211



2.06



2



4.12



69



6.0



C



F212



2.20



2



4.40



73



6.0



D



F214



2.23



2



4.45



74



6.0



D



F215



2.29



2



4.57



76


 

 



 



 



Mean



4.61



77



*           solvent control


LOQ:    1.22 µg test item /L


n.d.      = not detected


n.a.      = not applicable


The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.


 


Continued






































































































































































































































































































































Sampling Day



Nominal Concentration of Test Item
cnom



Repli-cate



Sample ID



Measured Concentration

x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d]



[µg/L]



 


 

[µg/L]


 

[µg/L]



[%]



14



0 (Control)



C



F222



n.d.



2



< LOQ



n.a.



0 (Control)*



C



F230



n.d.



2



< LOQ



n.a.



6.0



A



F235



1.87



2



3.74



62



6.0



A



F236



1.79



2



3.59



60



6.0



B



F238



1.63



2



3.27



54



6.0



B



F239



1.53



2



3.07



51



6.0



C



F241



1.60



2



3.21



53



6.0



C



F242



1.45



2



2.89



48



6.0



D



F244



1.59



2



3.18



53



6.0



D



F245



1.74



2



3.48



58


 

 



 



 



Mean



3.30



55



17



0 (Control)



D



F252



n.d.



2



< LOQ



n.a.



0 (Control)*



D



F260



n.d.



2



< LOQ



n.a.



6.0



A



F263



2.10



2



4.21



70



6.0



A



F264



1.99



2



3.99



66



6.0



B



F266



1.92



2



3.85



64



6.0



B



F267



1.93



2



3.87



64



6.0



C



F269



2.07



2



4.15



69



6.0



C



F270



1.94



2



3.89



65



6.0



D



F272



2.02



2



4.04



67



6.0



D



F273



1.96



2



3.92



65


 

 



 



 



Mean



3.99



66



19



0 (Control)



A



F276



n.d.



2



< LOQ



n.a.



0 (Control)*



A



F284



n.d.



2



< LOQ



n.a.



6.0



A



F293



2.09



2



4.19



70



6.0



A



F294



2.13



2



4.26



71



6.0



B



F296



2.17



2



4.34



72



6.0



B



F297



1.89



2



3.78



63



6.0



C



F299



1.88



2



3.75



63



6.0



C



F300



1.65



2



3.30



55



6.0



D



F302



2.44



2



4.88



81



6.0



D



F303



2.39



2



4.79



80


 

 



 



 



Mean



4.16



69



*           solvent control


LOQ:    1.22 µg test item /L


n.d.      = not detected


n.a.      = not applicable


The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.


 


 


Continued






































































































































































































































































































































Sampling Day



Nominal Concentration of Test Item
cnom



Repli-cate



Sample ID



Measured Concentration

x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d]



[µg/L]



 


 

[µg/L]


 

[µg/L]



[%]



21



0 (Control)



B



F307



n.d.



2



< LOQ



n.a.



0 (Control)*



B



F314



n.d.



2



< LOQ



n.a.



6.0



A



F321



2.03



2



4.06



68



6.0



A



F322



1.81



2



3.61



60



6.0



B



F324



1.72



2



3.44



57



6.0



B



F325



1.89



2



3.77



63



6.0



C



F327



1.76



2



3.52



59



6.0



C



F328



1.62



2



3.23



54



6.0



D



F330



2.26



2



4.52



75



6.0



D



F331



2.53



2



5.06



84


 

 



 



 



Mean



3.90



65



24



0 (Control)



C



F336



n.d.



2



< LOQ



n.a.



0 (Control)*



C



F344



n.d.



2



< LOQ



n.a.



6.0



A



F349



2.30



2



4.60



77



6.0



A



F350



2.03



2



4.06



68



6.0



B



F352



2.14



2



4.28



71



6.0



B



F353



2.10



2



4.20



70



6.0



C



F355



1.97



2



3.93



66



6.0



C



F356



1.94



2



3.88



65



6.0



D



F358



2.34



2



4.68



78



6.0



D



F359



2.20



2



4.40



73


 

 



 



 



Mean



4.25



71



26



0 (Control)



D



F368



n.d.



2



< LOQ



n.a.



0 (Control)*



D



F376



n.d.



2



< LOQ



n.a.



6.0



A



F379



2.15



2



4.30



72



6.0



A



F380



1.96



2



3.91



65



6.0



B



F382



2.03



2



4.06



68



6.0



B



F383



2.01



2



4.02



67



6.0



C



F385



2.05



2



4.10



68



6.0



C



F386



1.81



2



3.62



60



6.0



D



F388



2.20



2



4.41



73



6.0



D



F389



2.32



2



4.64



77


 

 



 



 



Mean



4.13



69



*           solvent control


LOQ     = 1.22 µg test item /L


n.d.      = not detected


n.a.      = not applicable


The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.


 


 


Continued






































































































































































































































































































































Sampling Day



Nominal Concentration of Test Item
cnom



Repli-cate



Sample ID



Measured Concentration

x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d]



[µg/L]



 


 

[µg/L]



 



[µg/L]



[%]



28



0 (Control)



A



F390



n.d.



2



< LOQ



n.a.



0 (Control)*



A



F398



n.d.



2



< LOQ



n.a.



6.0



A



F407



2.18



2



4.35



73



6.0



A



F408



2.14



2



4.28



71



6.0



B



F410



2.31



2



4.61



77



6.0



B



F411



2.18



2



4.36



73



6.0



C



F413



2.12



2



4.23



71



6.0



C



F414



2.18



2



4.37



73



6.0



D



F416



2.28



2



4.56



76



6.0



D



F417



2.16



2



4.31



72


 

 



 



 



Mean



4.39



73



31



0 (Control)



B



F420



n.d.



2



< LOQ



n.a.



0 (Control)*



B



F428



n.d.



2



< LOQ



n.a.



6.0



A



F435



1.68



2



3.37



56



6.0



A



F436



1.51



2



3.02



50



6.0



B



F438



2.18



2



4.36



73



6.0



B



F439



2.19



2



4.39



73



6.0



C



F441



1.53



2



3.06



51



6.0



C



F442



1.46



2



2.91



49



6.0



D



F444



1.82



2



3.64



61



6.0



D



F445



2.07



2



4.15



69


 

 



 



 



Mean



3.61



60



34



0 (Control)



C



F480



n.d.



2



< LOQ



n.a.



0 (Control)*



C



F488



n.d.



2



< LOQ



n.a.



6.0



A



F493



1.74



2



3.48



58



6.0



A



F494



1.60



2



3.21



53



6.0



B



F496



1.55



2



3.11



52



6.0



B



F497



1.76



2



3.52



59



6.0



C



F499



1.59



2



3.19



53



6.0



C



F500



1.84



2



3.68



61



6.0



D



F502



2.16



2



4.32



72



6.0



D



F503



2.01



2



4.01



67


 

 



 



 



Mean



3.57



59



*           solvent control


LOQ     = 1.22 µg test item /L


n.d.      = not detected


n.a.      = not applicable


The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.
Table 2: Results for application solutions from main test



























































































































Sampling Day / Age of Sample



Nominal Concentration of Test Item
cnom



Sample ID



Measured Concentration of Test Item
x



Sample Preparation Factor
F



Determined Concentration of Test Item
c



% of Nominal



[d/d]



[mg/L]


 

[µg/L]


 

[mg/L]



[%]



-2/0



60



App 44



3.20



15301



48.9



82



5/7



60



App 103



2.56



15301



39.2



65



5/0



60



App 102



2.92



15301



44.6



74



12/7



60



App 217



3.60



15301



55.2



92



12/0



60



App 216



3.06



15301



46.9



78



19/7



60



App 275



3.99



15301



61.1



102



19/0



60



App 274



3.60



15301



55.1



92



26/7



60



App 361



3.66



15301



56.1



93



26/0



60



App 360



3.68



15301



56.3



94



33/7



60



App 475



3.27



15301



50.0



83



33/0



60



App 474



4.04



15301



61.9



103



 


 


 


Table 3: Hatching success




































































































































































































































































































Treatment / Nominal
Test Item
Concentration


[µg/L]



Replicate



Larvae hatched/viable Eggs#
(20 Eggs per Replicate were inserted
at Test Start)



No of Larvae Hatched



Dead Eggs



Hatching Rate (HR)



Mean HR


± SD



Percent of


Solvent


Control



Day



0



1



2



3




 

 



[%]



[%]



[%]



Control



A



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



96.3


±


4.8



 


99


 


 n.s.*



B



0



/



20



0



/



20



0



/



20



1



/



18



18



/



0



18



2



90



C



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



2



/



18



19



/



0



19



1



95



 


Solvent


Control


 


 



A



0



/



20



0



/



20



0



/



20



1



/



19



19



/



0



19



1



95



 97.5


±


2.9



 


 



 



B



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



C



0



/



20



0



/



20



0



/



20



2



/



18



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



1



/



18



19



/



0



19



1



95



 


6.0


 ( 4.0 ) + 


 



A



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



 98.8


±


2.5



 


101 


 n.s.



B



0



/



20



0



/



20



0



/



20



4



/



16



20



/



0



20



0



100



C



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



3



/



17



19



/



0



19



1



95



 


#:            Dead eggs are not included in this counting. Eggs which were missing were considered to be dead.


°:            Day 4 was determined to be the end of the hatching period (all larvae hatched in the control and solvent control)


+:               Mean measured test item concentration


SD:         Standard deviation


n.s.*:       Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.:        Not statistically significantly different compared to the solvent control according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


 


 


Table 4: Survival of larvae and juvenile fish







































































































































































































































































Treatment / Nominal


Test Item


Concentration






[µg/L]



Replicate



No. of Larvae
hatched at Day 4



Mortality
(No. of Dead Fish)



Dead Fish at Test End



Surviving Fish



Survival Rate [%]



Mean Survival Rate [%]
± SD



Percent of Solvent Control [%]



Day of the Test



5



7



11



13



18



20



24



28



31



34



(Day 0 Post Hatch equals Day 4 Post Fertilization)



Day Post Hatch



1



3



7



9



14



16



20



24



26



29



Control



A



20



 



1



 



2



 



 



 



 



 



 



3



17



85



 


87.0


±


2.8



97.0


 


n.s.#



B



18



1



1



 



 



 



 



 



 



 



 



2



16



89



C



20



 



1



 



1



 



 



 



 



 



 



2



18



90



D



19



 



 



1



2



 



 



 



 



 



 



3



16



84



 


Solvent Control


 


 



A



19



 



1



1



 



 



1



 



 



 



 



3



16



84



 


89.7


±


9.2



-



B



20



 



1



1



2



 



 



 



 



 



 



4



16



80



C



20



 



 



 



 



 



 



 



 



 



 



0



20



100



D



19



 



 



 



 



 



1



 



 



 



 



1



18



95



 


6.0


 ( 4.0 ) +


 



A



20



 



 



1



1



 



 



 



 



 



 



2



18



90



 


87.4


±


2.7



97.4
n.s.



B



20



1



1



 



 



1



 



 



 



 



 



3



17



85



C



20



 



 



1



 



2



 



 



 



 



 



3



17



85



D



19



2



 



 



 



 



 



 



 



 



 



2



17



89



 


SD:      Standard deviation


+:               Mean measured test item concentration


n.s.*:       Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.:        Not statistically significantly different compared to the solvent control according to a Fisher’s Exact Binominal test, one-sided smaller, α = 0.05.


 


 


 


 


 


 


 


 


 


Table 5: Body length of the surviving fish at the end of the test











































































































































































































































































































































































Treatment / Nominal
Test Item Concentration


 


[µg/L]



Replicate



 Body Lenght of Each Individual Test Fish



[mm]



Number of Fish




Mean


per Repli-cate
[mm]



 Mean


per


Treatment
± SD


[mm]



Percent


of


Solvent


Control
[%]



Control



A



21



20



22



20



21



17



22



21



21



18



17



20.1



 



100


 


n.s.#



20



18



20



22



18



20



20



 



 



 



20.0



B



20



22



21



20



20



21



20



20



20



21



16



20.4



20



22



19



20



20



21



 



 



 



 



±



C



21



19



19



21



19



20



16



20



19



21



18



19.9



0.3



21



20



21



20



21



19



21



20



 



 



D



16



19



19



19



21



21



21



20



20



20



16



19.8



 



20



20



20



19



21



20



 



 



 



 



 



 


 


 


Solvent Control


 


 


 



A



21



21



20



19



19



21



20



21



21



21



16



20.1



 



-



20



20



19



20



18



21



 



 



 



 



20.0



B



19



20



22



21



21



20



19



20



20



19



16



20.3



20



21



21



18



21



22



 



 



 



 



±



C



20



21



21



21



19



21



21



20



19



20



20



20.0



0.4



20



20



20



20



20



20



20



18



20



19



D



19



17



20



20



20



20



18



20



20



20



18



19.4



 



18



20



20



19



19



20



20



20



 



 



 



 


 


6.0


( 4.0 ) +


 



A



20



20



20



21



20



21



21



22



18



21



18



19.7



 



101


 


n.s.°



20



20



20



21



18



14



21



17



 



 



20.1



B



20



22



22



20



19



19



21



18



22



19



17



20.3



21



18



20



21



22



21



20



 



 



 



±



C



22



18



22



20



20



21



21



20



21



21



17



20.3



0.3



21



19



20



21



21



21



16



 



 



 



D



20



21



20



20



19



21



21



22



20



20



17



20.2



 



20



21



21



18



20



20



19



 



 



 



 



 


SD:      Standard deviation


+:           Mean measured test item concentration


n.s.*:       Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.°:       Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


 


Table 6: Fish wet weight at the end of the test











































































































































































































































































































































































Treatment / Nominal Test Item Concentration


 


[µg/L]



Replicate



 Wet Weight of Each Individual Test Fish

[mg]



Number of Fish



Mean


per Replicate

[mg]



 Mean


per


Treat-ment
± SD


[mg]



Percent


of Solvent Control
[%]



Control



A



62



50



78



65



73



43



73



70



71



46



17



62.2



 



 


100


 


n.s.#


 



58



50



64



79



48



69



59



 



 



 



61.0



B



62



73



66



62



57



57



62



55



62



61



16



62.3



58



72



50



61



67



71



 



 



 



 



±



C



74



57



52



72



58



65



30



51



47



72



18



60.9



10.1



64



52



71



57



79



55



78



63



 



 



D



34



55



53



55



72



69



66



56



61



57



16



58.4



 



62



59



61



48



69



58



 



 



 



 



 



 


 


 


Solvent


Control


 


 


 



A



72



73



59



58



55



75



63



82



74



66



16



65.7



 



 


 -


 


 


 


 



66



60



61



67



53



67



 



 



 



 



61.0



B



55



68



76



73



76



56



51



69



65



49



16



65.8



73



76



70



45



74



76



 



 



 



 



±



C



61



70



74



64



57



66



64



55



50



60



20



59.1



5.9



60



57



64



59



59



55



57



46



56



48



D



53



42



52



51



61



53



35



54



55



58



18



53.5



 



45



68



55



54



50



58



56



63



 



 



 



 


 


6.0


( 4.0 ) +


 



A



54



70



59



74



66



71



64



74



47



64



18



60.1



 



 


 


 


105


 


n.s.


 


 



57



66



64



73



45



22



66



45



 



 



63.8



B



68



67



68



60



54



45



77



43



84



52



17



64.3



73



45



65



65



88



73



66



 



 



 



±



C



80



41



81



63



59



72



74



67



73



88



17



66.4



2.7



72



51



70



73



68



70



27



 



 



 



D



61



78



63



61



60



69



78



85



62



63



17



64.5



 



58



70



72



51



63



55



48



 



 



 



 



 


SD:      Standard deviation


+:         Mean measured test item concentration


n.s.#:    Control and solvent control are not statistically significantly different, according to a Student t-test, two-sided, α = 0.05.


n.s.:     Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


 


Table 7: Fish dry weight at the end of the test

































































































































Treatment / Nominal Test Item Concentration


 


[µg/L]



Replicate



Fish Dry Weight per Replicate



[mg]



Number of Surviving Fish



Mean Fish Weight per Replicate
[mg]



Min



[mg]



Max



[mg]



Mean ± SD Fish Dry Weight Per Treatment



[mg]



% of Solvent Control


 
 
 

Control



A



285.3



17



16.78



15.68



16.89



16.43



±



0.55



100.5


 


n.s.*


 

B



270.2



16



16.89


 

C



295.0



18



16.39


 

D



250.9



16



15.68


 

Solvent Control



A



281.4



16



17.59



14.19



18.01



16.35



±



1.78




 

B



288.2



16



18.01


 

C



312.4



20



15.62


 

D



255.5



18



14.19


 

6.0
( 4.0 )+



A



291.4



18



16.19



16.19



18.01



17.27



±



0.79



105.6


 


n.s.


 

B



300.5



17



17.68


 

C



306.1



17



18.01


 

D



292.4



17



17.20


 

 


SD:      Standard deviation


+:         Mean measured test item concentration


n.s.*:    Control and solvent control are not statistically significantly different, according to a Student t-test, two-sided, α = 0.05.


n.s.:     Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


Table 8: Dissolved Oxygen Concentrations in the Test Medium and the Controls
























































































































































































































































































































Exposure Time

[Days]



Replicate





Dissolved Oxygen Concentrations [mg/L]



Treatment / Nominal Test Item Concentration [µg/L]



Control



Solvent Control



6.0



0



A



7.8



7.8



7.8



B



7.8



7.8



7.8



C



7.8



7.8



7.8



D



7.8



7.8



7.8



5



A



7.6



7.6



7.6



B



7.6



7.5



7.5



C



7.7



7.6



7.5



D



7.6



7.6



7.6



7



A



7.5



7.4



7.4



B



7.4



7.4



7.3



C



7.3



7.3



7.3



D



7.5



7.5



7.4



11



A



7.4



7.3



7.3



B



7.4



7.2



7.3



C



7.3



7.1



7.2



D



7.4



7.2



7.2



14



A



7.6



7.5



7.5



B



7.5



7.5



7.4



C



7.6



7.4



7.5



D



7.6



7.5



7.5



18



A



7.8



7.8



7.8



B



7.8



7.7



7.8



C



7.8



7.7



7.8



D



7.8



7.7



7.8



20



A



7.7



7.6



7.8



B



7.6



7.7



7.7



C



7.6



7.7



7.6



D



7.7



7.5



7.6



24



A



7.6



7.7



7.7



B



7.6



7.7



7.7



C



7.8



7.8



7.8



D



7.8



7.8



7.7



28



A



7.3



7.2



7.5



B



7.2



7.1



7.5



C



7.2



7.2



7.4



D



7.2



7.1



7.5



32



A



7.7



7.6



7.5



B



7.7



7.5



7.5



C



7.6



7.5



7.5



D



7.6



7.5



7.5



34



A



7.7



7.6



7.6



B



7.7



7.6



7.6



C



7.7



7.6



7.6



D



7.6



7.6



7.6



Minimum



7.2



7.1



7.2



Maximum



7.8



7.8



7.8



Mean



7.6



7.5



7.6



 


 


Table 9: Summary of biological results




















































Biological Endpoints



Biological Endpoints
Based on Mean Measured Test Item Concentration



 



Control




Solvent Control



4.0 µg/L
[6.0 µg/L*]



% of
Solvent Control



Hatching Success [%] after 4 Days of Exposure



96



98



99 (n.s.)



102 %



Survival [%] after 34 Days of Exposure



87



90



87 (n.s.)



97 %



Mean Fish Body Length [mm] after 34 Days of Exposure



20.0



20.0



20.1 (n.s.)



101 %



Mean Fish Wet Weight [mg]
after 34 Days of Exposure



61.0



61.0



63.8 (n.s.)



105 %



Mean Fish Dry Weight per Treatment [mg] after 34 Days of Exposure



16.4



16.4



17.3 (n.s.)



106 %



n.s.:   Not statistically significantly different from the solvent control


*:        Nominal concentration


 


 


Table 10: Hatching Success




































































































































































































































































































Treatment / Nominal
Test Item
Concentration


[µg/L]



Replicate



Larvae hatched/viable Eggs#
(20 Eggs per Replicate were inserted
at Test Start)



No of Larvae Hatched



Dead Eggs



Hatching Rate (HR)



Mean HR


± SD



Percent of


Solvent


Control



Day



0



1



2



3




 

 



[%]



[%]



[%]



Control



A



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



96.3


±


4.8



 


99


 


 n.s.*



B



0



/



20



0



/



20



0



/



20



1



/



18



18



/



0



18



2



90



C



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



2



/



18



19



/



0



19



1



95



 


Solvent


Control


 


 



A



0



/



20



0



/



20



0



/



20



1



/



19



19



/



0



19



1



95



 97.5


±


2.9



 


 



 



B



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



C



0



/



20



0



/



20



0



/



20



2



/



18



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



1



/



18



19



/



0



19



1



95



 


6.0


 ( 4.0 ) + 


 



A



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



 98.8


±


2.5



 


101 


 n.s.



B



0



/



20



0



/



20



0



/



20



4



/



16



20



/



0



20



0



100



C



0



/



20



0



/



20



0



/



20



1



/



19



20



/



0



20



0



100



D



0



/



20



0



/



20



0



/



20



3



/



17



19



/



0



19



1



95



 #: Dead eggs are not included in this counting. Eggs which were missing were considered to be dead.


°: Day 4 was determined to be the end of the hatching period (all larvae hatched in the control and solvent control)


+: Mean measured test item concentration


SD: Standard deviation


n.s.*: Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.: Not statistically significantly different compared to the solvent control according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


 


Table 11: Survival of Larvae and Juvenile Fish







































































































































































































































































Treatment / Nominal


Test Item


Concentration






[µg/L]



Replicate



No. of Larvae
hatched at Day 4



Mortality
(No. of Dead Fish)



Dead Fish at Test End



Surviving Fish



Survival Rate [%]



Mean Survival Rate [%]
± SD



Percent of Solvent Control [%]



Day of the Test



5



7



11



13



18



20



24



28



31



34



(Day 0 Post Hatch equals Day 4 Post Fertilization)



Day Post Hatch



1



3



7



9



14



16



20



24



26



29



Control



A



20



 



1



 



2



 



 



 



 



 



 



3



17



85



 


87.0


±


2.8



97.0


 


n.s.#



B



18



1



1



 



 



 



 



 



 



 



 



2



16



89



C



20



 



1



 



1



 



 



 



 



 



 



2



18



90



D



19



 



 



1



2



 



 



 



 



 



 



3



16



84



 


Solvent Control


 


 



A



19



 



1



1



 



 



1



 



 



 



 



3



16



84



 


89.7


±


9.2



-



B



20



 



1



1



2



 



 



 



 



 



 



4



16



80



C



20



 



 



 



 



 



 



 



 



 



 



0



20



100



D



19



 



 



 



 



 



1



 



 



 



 



1



18



95



 


6.0


 ( 4.0 ) +


 



A



20



 



 



1



1



 



 



 



 



 



 



2



18



90



 


87.4


±


2.7



97.4
n.s.



B



20



1



1



 



 



1



 



 



 



 



 



3



17



85



C



20



 



 



1



 



2



 



 



 



 



 



3



17



85



D



19



2



 



 



 



 



 



 



 



 



 



2



17



89



SD: Standard deviation


+: Mean measured test item concentration


n.s.*: Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.: Not statistically significantly different compared to the solvent control according to a Fisher’s Exact Binominal test, one-sided smaller, α = 0.05


 


 


Table 12: Body Length of the Surviving Fish at the End of the Test











































































































































































































































































































































































Treatment / Nominal
Test Item Concentration


 


[µg/L]



Replicate



 Body Lenght of Each Individual Test Fish



[mm]



Number of Fish




Mean


per Repli-cate
[mm]



 Mean


per


Treatment
± SD


[mm]



Percent


of


Solvent


Control
[%]



Control



A



21



20



22



20



21



17



22



21



21



18



17



20.1



 



100


 


n.s.#



20



18



20



22



18



20



20



 



 



 



20.0



B



20



22



21



20



20



21



20



20



20



21



16



20.4



20



22



19



20



20



21



 



 



 



 



±



C



21



19



19



21



19



20



16



20



19



21



18



19.9



0.3



21



20



21



20



21



19



21



20



 



 



D



16



19



19



19



21



21



21



20



20



20



16



19.8



 



20



20



20



19



21



20



 



 



 



 



 



 


 


 


Solvent Control


 


 


 



A



21



21



20



19



19



21



20



21



21



21



16



20.1



 



-



20



20



19



20



18



21



 



 



 



 



20.0



B



19



20



22



21



21



20



19



20



20



19



16



20.3



20



21



21



18



21



22



 



 



 



 



±



C



20



21



21



21



19



21



21



20



19



20



20



20.0



0.4



20



20



20



20



20



20



20



18



20



19



D



19



17



20



20



20



20



18



20



20



20



18



19.4



 



18



20



20



19



19



20



20



20



 



 



 



 


 


6.0


( 4.0 ) +


 



A



20



20



20



21



20



21



21



22



18



21



18



19.7



 



101


 


n.s.°



20



20



20



21



18



14



21



17



 



 



20.1



B



20



22



22



20



19



19



21



18



22



19



17



20.3



21



18



20



21



22



21



20



 



 



 



±



C



22



18



22



20



20



21



21



20



21



21



17



20.3



0.3



21



19



20



21



21



21



16



 



 



 



D



20



21



20



20



19



21



21



22



20



20



17



20.2



 



20



21



21



18



20



20



19



 



 



 



 



SD:      Standard deviation


+:           Mean measured test item concentration


n.s.*:       Control and solvent control are not statistically significantly different, according to a Fisher’s Exact Binominal test, two-sided, α = 0.05.


n.s.°:       Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


 


Table 13: Fish Wet Weight at the End of the Test











































































































































































































































































































































































Treatment / Nominal Test Item Concentration


 


[µg/L]



Replicate



 Wet Weight of Each Individual Test Fish

[mg]



Number of Fish



Mean


per Replicate

[mg]



 Mean


per


Treat-ment
± SD


[mg]



Percent


of Solvent Control
[%]



Control



A



62



50



78



65



73



43



73



70



71



46



17



62.2



 



 


100


 


n.s.#


 



58



50



64



79



48



69



59



 



 



 



61.0



B



62



73



66



62



57



57



62



55



62



61



16



62.3



58



72



50



61



67



71



 



 



 



 



±



C



74



57



52



72



58



65



30



51



47



72



18



60.9



10.1



64



52



71



57



79



55



78



63



 



 



D



34



55



53



55



72



69



66



56



61



57



16



58.4



 



62



59



61



48



69



58



 



 



 



 



 



 


 


 


Solvent


Control


 


 


 



A



72



73



59



58



55



75



63



82



74



66



16



65.7



 



 


 -


 


 


 


 



66



60



61



67



53



67



 



 



 



 



61.0



B



55



68



76



73



76



56



51



69



65



49



16



65.8



73



76



70



45



74



76



 



 



 



 



±



C



61



70



74



64



57



66



64



55



50



60



20



59.1



5.9



60



57



64



59



59



55



57



46



56



48



D



53



42



52



51



61



53



35



54



55



58



18



53.5



 



45



68



55



54



50



58



56



63



 



 



 



 


 


6.0


( 4.0 ) +


 



A



54



70



59



74



66



71



64



74



47



64



18



60.1



 



 


 


 


105


 


n.s.


 


 



57



66



64



73



45



22



66



45



 



 



63.8



B



68



67



68



60



54



45



77



43



84



52



17



64.3



73



45



65



65



88



73



66



 



 



 



±



C



80



41



81



63



59



72



74



67



73



88



17



66.4



2.7



72



51



70



73



68



70



27



 



 



 



D



61



78



63



61



60



69



78



85



62



63



17



64.5



 



58



70



72



51



63



55



48



 



 



 



 



SD: Standard deviation


+: Mean measured test item concentration


n.s.#: Control and solvent control are not statistically significantly different, according to a Student t-test, two-sided, α = 0.05.


n.s.: Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


 


Table 14: Fish Dry Weight at the End of the Test

































































































































Treatment / Nominal Test Item Concentration


 


[µg/L]



Replicate



Fish Dry Weight per Replicate



[mg]



Number of Surviving Fish



Mean Fish Weight per Replicate
[mg]



Min



[mg]



Max



[mg]



Mean ± SD Fish Dry Weight Per Treatment



[mg]



% of Solvent Control


 
 
 

Control



A



285.3



17



16.78



15.68



16.89



16.43



±



0.55



100.5


 


n.s.*


 

B



270.2



16



16.89


 

C



295.0



18



16.39


 

D



250.9



16



15.68


 

Solvent Control



A



281.4



16



17.59



14.19



18.01



16.35



±



1.78




 

B



288.2



16



18.01


 

C



312.4



20



15.62


 

D



255.5



18



14.19


 

6.0
( 4.0 )+



A



291.4



18



16.19



16.19



18.01



17.27



±



0.79



105.6


 


n.s.


 

B



300.5



17



17.68


 

C



306.1



17



18.01


 

D



292.4



17



17.20


 

 


SD:      Standard deviation


+:         Mean measured test item concentration


n.s.*:    Control and solvent control are not statistically significantly different, according to a Student t-test, two-sided, α = 0.05.


n.s.:     Not statistically significantly different compared to solvent control, according to a Two-sample t-test, two-sided, α = 0.05.


 


 


 


VALIDITY OF STUDY


The test was considered to be valid as the following criteria given by the test guideline were fulfilled:



  • The dissolved oxygen concentration in the test medium should be at least 60% of the air saturation value and was at least 92 % throughout the test.

  • The water temperature should not differ by more than ±1.5 °C between the aquaria or between successive days at any one time during the test. In the test, differences in water temperature did not exceed 0.3°C.

  • Hatching success in the control should be higher than 70% and was 96.3 and 97.5 % in the control and solvent control, respectively. Post-hatch success should be at least 75% and was 87 and 89.7 % in the control and solvent control, respectively.

Validity criteria fulfilled:
yes
Conclusions:
In a Fish Early Life Stage Test with Danio rerio according to OECD TG 210 at the limit concentration of 4.0 µg/L (arithm. mean measured, corresponding to solubility limit of test item in test medium), the overall NOEC of the test item was determined to be equal to or above 4.0 μg/L (arithm. mean measured), as no adverse effects were observed on hatching, survival and growth of the fish. The LOECs and EC10 values for the different endpoints were > 4.0 μg/L (arithm. mean measured). In conclusion, the test item had no toxic effects on early life stages of Danio rerio over the exposure period of 34 days at the applied limit concentration, which corresponds to the solubility limit of the test item in the test medium.
Executive summary:

The toxicity of the test item to zebra fish (Danio rerio) was investigated in an early-life stage toxicity test according to the OECD Guideline for Testing of Chemicals, No. 210, "Fish, Early-life Stage Toxicity Test", 2013, and in compliance with GLP principles. A limit test under flow-through conditions was performed to demonstrate that the test item has no toxic effects on zebra fish at the highest concentration of the test item that could be reached in test water. The nominal test item concentration was 6.0 μg/L. A control group and a solvent control group were tested in parallel. The test duration was 34 days. At the start of the test, 80 eggs (divided into 4 replicates) were introduced in the test for the test concentration, the control and solvent control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival. The test concentration selected for the main test was based on two pre-experiments to the solubility limit of the test item in test water. The maximum solubility was found to be in the range of 1.1 to 4.6 μg/L, based on several measurements of the dissolved fraction of the test item in equilibrated suspensions with a loading rate of 100 mg/L. From these results, the maximum solubility of the test item in test water was estimated to be approximately 2.1 μg/L. During two dosing pre-experiments in the flow-through system it became evident, that the recoveries of the test concentrations in the test media were approximately 50%. The losses were attributed to intrinsic properties of the difficult test item with low water solubility. To compensate these losses, a nominal concentration of 6.0 μg/L was chosen for the main test to maintain a constant exposure concentration at the solubility limit of the test item in test water of approximately 2 to 3 μg/L. The test was run under flow-through conditions with an at least 10-fold test medium renewal rate per day. The solvent N,N-dimethylformamide p.a. (DMF) was used for the preparation of a concentrated application solution, which was used for the dosage of the test item in test water. The final concentration of the solvent in the test medium was 50 μL/L. The concentrations of the test item in the test media were analytically determined in duplicate samples from all four replicates of the test concentration at the start of the test and at 14 further sampling days. The mean measured concentrations for each sampling day (mean of four replicates) were in the range of 55 to 77 % of the nominal value. The mean concentrations for each sampling day (mean of the four replicates) were in the range of ± 20 % of the overall mean measured concentration. The overall mean measured concentration of the test item over the test period of 34 days was calculated as arithmetic mean of all measurements of the test concentration and was 4.0 μg/L, corresponding to 66 % of the nominal value. This recovery is in the range of what was expected according to the dosing pre-experiments. The measured concentration of 4.0 μg/L corresponded to the solubility limit of the test item in test water of approximately 2 to 3 μg/L.


As a result, the test item had no toxic effects on early life stages of zebra fish (Danio rerio) over the exposure period of 34 days at the limit test concentration of mean measured 4.0 μg/L. Therefore, considering the NOECs for each of the test parameters assessed, the overall NOEC of the test item for early life stages of zebra fish was determined to be equal to or above the water solubility limit of 4.0 μg/L (mean measured), as no adverse effects were observed on hatching, survival and growth of the fish at this test item concentration. The LOECs and EC10 values for the different endpoints were > 4.0 μg/L. All validity criteria of the guideline were fulfilled. The test is thus regarded valid and acceptable for assessment.

Description of key information

In a Fish Early Life Stage Test with Danio rerio according to OECD TG 210 at the limit concentration of 4.0 µg/L (arithm. mean measured, corresponding to solubility limit of test item in test medium), the overall NOEC of the test item was determined to be equal to or above 4.0 µg/L (arithm. mean measured), as no adverse effects were observed on hatching, survival and growth of the fish. The LOECs and EC10 values for the different endpoints were > 4.0 µg/L (arithm. mean measured). In conclusion, the test item had no toxic effects on early life stages of Danio rerio over the exposure period of 34 days at the applied limit concentration, which corresponds to the solubility limit of the test item in the test medium.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
4 µg/L

Additional information

The toxicity of the test item to zebra fish (Danio rerio) was investigated in an early-life stage toxicity test according to the OECD Guideline for Testing of Chemicals, No. 210, "Fish, Early-life Stage Toxicity Test", 2013, and in compliance with GLP principles. A limit test under flow-through conditions was performed to demonstrate that the test item has no toxic effects on zebra fish at the highest concentration of the test item that could be reached in test water. The nominal test item concentration was 6.0 μg/L. A control group and a solvent control group were tested in parallel. The test duration was 34 days. At the start of the test, 80 eggs (divided into 4 replicates) were introduced in the test for the test concentration, the control and solvent control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival. The test concentration selected for the main test was based on two pre-experiments to the solubility limit of the test item in test water. The maximum solubility was found to be in the range of 1.1 to 4.6 μg/L, based on several measurements of the dissolved fraction of the test item in equilibrated suspensions with a loading rate of 100 mg/L. From these results, the maximum solubility of the test item in test water was estimated to be approximately 2.1 μg/L. During two dosing pre-experiments in the flow-through system it became evident, that the recoveries of the test concentrations in the test media were approximately 50%. The losses were attributed to intrinsic properties of the difficult test item with low water solubility. To compensate these losses, a nominal concentration of 6.0 μg/L was chosen for the main test to maintain a constant exposure concentration at the solubility limit of the test item in test water of approximately 2 to 3 μg/L. The test was run under flow-through conditions with an at least 10-fold test medium renewal rate per day. The solvent N,N-dimethylformamide p.a. (DMF) was used for the preparation of a concentrated application solution, which was used for the dosage of the test item in test water. The final concentration of the solvent in the test medium was 50 μL/L. The concentrations of the test item in the test media were analytically determined in duplicate samples from all four replicates of the test concentration at the start of the test and at 14 further sampling days. The mean measured concentrations for each sampling day (mean of four replicates) were in the range of 55 to 77 % of the nominal value. The mean concentrations for each sampling day (mean of the four replicates) were in the range of ± 20 % of the overall mean measured concentration. The overall mean measured concentration of the test item over the test period of 34 days was calculated as arithmetic mean of all measurements of the test concentration and was 4.0 μg/L, corresponding to 66 % of the nominal value. This recovery is in the range of what was expected according to the dosing pre-experiments. The measured concentration of 4.0 μg/L corresponded to the solubility limit of the test item in test water of approximately 2 to 3 μg/L.


 


As a result, the test item had no toxic effects on early life stages of zebra fish (Danio rerio) over the exposure period of 34 days at the limit test concentration of mean measured 4.0 μg/L. Therefore, considering the NOECs for each of the test parameters assessed, the overall NOEC of the test item for early life stages of zebra fish was determined to be equal to or above the water solubility limit of 4.0 μg/L (mean measured), as no adverse effects were observed on hatching, survival and growth of the fish at this test item concentration. The LOECs and EC10 values for the different endpoints were > 4.0 μg/L. All validity criteria of the guideline were fulfilled. The test is thus regarded valid and acceptable for assessment.