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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:
12/11/2021 - 16/12/2021
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
See "any other information on materials" for details
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test Item name 1-ethoxy-2-(2-methoxyethoxy)ethane
Lot number 09511
Active ingredient content: ˃ 99.9%
Appearance water clear, liquid
Expiry date March 28, 2023
Storage at room temperature, protect from light
Analytical monitoring:
yes
Remarks:
Analytical control of the test solutions was performed with a validated method (Validation Study No.: 103-100-6044) by the Analytical Laboratory of TOXI-COOP ZRT. The samples were analysed GC-MS method.
Details on sampling:
The selected nominal concentrations (spaced by a
factor of 2.3) were 0.36 mg/L, 0.82 mg/L, 1.9 mg/L, 4.3 mg/L and
10 mg/L which are the same as was used in the Daphnia magna 21-day
reproduction test performed previously in the testing laboratory
(Study No.: 103-211-6041).
For determination of 1-ethoxy-2-(2-methoxyethoxy)ethane
concentration samples were taken from the test solutions and from the
negative control at the start and at the end of the renewal periods. Test
item concentrations were analysed in the test solutions by a GC-MS
method. 1-ethoxy-2-(2-methoxyethoxy)ethane concentration was
determined at 9 renewal periods.


During the test, the concentration of the test item in the test solutions were determined at
regular intervals to characterise exposure. Both the freshly prepared test solutions (“new”
test solutions) and the test solutions removed from the test chambers (“old” test solutions)
before use of the “new” solutions were analysed regularly.
Determinations were made for 9 renewal periods. Both “new” and “old” test solutions were
analysed in all separate test chambers at each occasion.
Samples were taken on the following days: 0, 4, 7, 11, 14, 18, 21, 25, 31 and 34.
Vehicle:
not specified
Details on test solutions:
The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent, as the test item was well water soluble. A stock solution was first prepared by dissolving an appropriate amount of test item in the dilution water, then the test solutions were prepared by appropriate dilutions of this stock solution. Once the test solutions prepared the appropriate volume was used for transfer of the test animals and the remaining test solutions were stored under ambient temperature (at the same place as the test aquariums) and used as necessary for renewal of the test solutions in the test aquariums (by replenishment or transfer,)

Control

A dilution water control was run in addition to the test item concentration series.

Table 3. Preparation of the Stock and Test Solutions see "any other information on results".
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebrafish (Danio rerio)
Origin: The breeding stocks used for production of eggs is obtained from
Toma István e.v.
Hungary, 2146 Mogyoród, Gödöllői u. 41/B. and
from Hungarian University of Agriculture and Life Sciences Hungary, 2103 Gödöllő, Páter K. u. 1.
Number of eggs / test groups: 100 (divided equally between the replicates)
Number of replicates / test groups: 4
Stage of eggs at start of exposition: The embryonic stage at the beginning of exposure was verified as much as possible: stage of embryonic development at the time of pre-treatment was approximately 2-32 cells in the test solutions and the control.
Test type:
semi-static
Water media type:
other: Reconstituted water diluted with purified water in a ratio of 3 : 2 (3 part reconstituted water: 2 part purified water) was used as dilution water in the test. see "any other information on results"
Limit test:
no
Total exposure duration:
34 d
Hardness:
see "any other information on results"
Test temperature:
26 ± 1.5 °C
see "any other information on results"
pH:
see "any other information on results"
Dissolved oxygen:
60.7 % – 95.4 % air saturation value
see "any other information on results"
Test solutions were slightly aerated from Day 5 to Day 24 (during the period when dry food was provided)
Salinity:
not specified in study report
Conductivity:
not specified in study report
Nominal and measured concentrations:
see Table 8 Summarized Results of Analytical Determination of the Test Concentrations (Determined for 9 Renewal Periods) in "any other information on results"
Details on test conditions:
Test Chambers

Type and Size: Suitable sized glass Petri-dishes with 25-30 mL test solutions (until free-feeding stage);
Suitable sized glass aquaria with 0.25-0.5 L test solutions (from free-feeding stage to the end of the test).
Identification: Each test unit was uniquely identified with study number, test item concentration and number of parallels.
Allocation: Until free-feeding stage the test chambers were kept in a temperature-controlled incubator. The free-feeding larvae were transferred to suitable sized test aquariums and kept on a temperature-controlled tray until end of the test.

Environmental Conditions

Temperature: The temperature was maintained within a range of 26 ± 1.5 °C and was appropriately constant (the water temperature could not differ by more than ± 1.5 °C between the test chambers or between the successive days at any time during the test).
Photoperiod: 16 h light: 8 h dark
Oxygen concentration: Test solutions were not aerated until start of feeding (from Day 0 to Day 4). After start of feeding of the larvae, test solutions were slightly aerated during the period when dry food was given (from Day 5 to Day 24). No aeration was used from Day 25 to Day 34.

Duration of the Test

The test started as soon as it was technically possible after the eggs have been fertilized and was continued 30 days post-hatch of the control fish.

Replenishment and Renewal of the Test Solutions, Transfer of the Test Organisms

Renewal of the test solutions was performed by transfer of the test organisms from “old” test solutions to “new” test solutions. Replenishment of the test solutions (lost because of removal of uneaten food and faces) was also performed.
For renewal a new batch of test solutions were prepared. Within the stability period (i. e. 7 days; determined during the analytical method validation) these test solutions were used as necessary for replenishment or for transfer of the test organisms.
Days of preparation of a new batches of test solutions: Days 0, 4, 7, 11, 14, 18, 21, 25 and 31.
Days of transfer of test animals: from Day 4 to Day 25 and on Day 31 (fish were transferred in the afternoon before feeding)
Days of replenishment of the test solutions (without transfer of the test animals): - once a day at the observation: from Day 0 to Day 3
- once a day in the morning, before the feeding: from Day 5 to Day 25 and on Day 31 - twice a day before the feedings: from Day 26 to Day 30 and on Days 32, 33.

Loading

One hundred eggs, divided equally between the four replicates, were used per test concentrations and the control. Volume of the test solutions was increased from 25-30 mL to 250 mL (on Day 4) and finally to 500 mL (from Day 5 to Day 34) to maintain loading of the test chambers as low as technically possible during the test.

Feeding

The fish were fed from elimination of yolk sac (free-feeding stage) to the end of the test twice a day with appropriate type and amount of food (except on the first feeding day when food was supplied once only).
Dry food (fine powder) was introduced in the afternoon of Day 4. Live food (Artemia naupli; AN) was introduced on Day 14. From Day 14 to Day 24 both type of food (DF and AN) was supplied to the animals. Fish were not fed on the day of termination of the test (Day 34). Both type of food was supplied ad libitum (means supplying excessive amount of food for the test organisms regularly).
Dry food (DF) was weighed and suspended in the dilution water at each occasion.
On Day 4 0.35 g dry food was suspended in 14 ml dilution water and same volume (0.5 mL) of this suspension was supplied for each test aquarium (in the afternoon only) meaning 12.5 mg dry food/test aquarium/day.
From Day 5 to Day 20 0.35 g dry food was suspended in 14 ml dilution water and same volume (0.5 mL) of this suspension was supplied for each test aquarium twice a day meaning 25.0 mg dry food/test aquarium/day.
From Day 21 to Day 24 0.175 g dry food was suspended in 7 mL dilution water and 0.25 mL of this suspension was supplied for each test aquarium twice a day meaning
12.5 mg dry food/test aquarium/day.
For producing live food (AN) brine shrimp eggs (approximately 1.5 mL) were placed into 500 mL salt water (6.25 g or 10 g NaCl/500 mL dilution water) and left to hatch for approximately 24 or 36 hours. Two parallels were prepared in the morning of each day. Hatched nauplies (one portion in the morning and one in the afternoon) were filtered and suspended in 14 mL dilution water and 0.5 mL of this suspension was supplied for each test aquarium twice a day from Day 14 to Day 33.
Dry food used in the test:
Name: Sera Micron Nature Appearance: Fine powder
Main components: Spirulina (51 %), krill (18 %), Artemia Shrimp, Omega fatty acids
(34%), fish flour, wheat flour, Brewer’s yeast
Manufacturer: Sera, D-52518
Heinsberg, Germany
Storage: 2-8 °C
Identification Code: SM210712/1 and SM210712/2
Charge No.: 0000077382 Expiry date: 01/2024

Live food (Artemia nauplii) prepared from:
Name: Artemio®Pur
Artemia (brine shrimp) eggs
Manufacturer: JBL GmbH & Co. KG, D-67141
Neuhofen, Germany
Storage: 2-8 °C
Identification Code: AP210712/1 and AP210712/2
Expiry date: 03/2023
Feeding regime is summarized in Table 24 see "any other information on results".

Cleaning

Surplus food and faces were removed by syphoning before replenishment of the test solutions (in the mornings) and by transfer of the test animals into a clean test aquarium (in the afternoons).

Water Quality Measurements

The water temperature was determined at least once a day in all test chambers. In addition, water temperature was continuously monitored with a min/max thermometer in a concurrent test chamber containing the dilution water only at the same volume as the test solutions in the test chambers. Measured temperature values are given in Table 16 and Table 17 see "any other information on results" .
Dissolved oxygen concentrations (LDO) and pH were determined at least weekly in all test chambers.
LDO was measured on Days 0, 4, 5, 6, 7, 8, 11, 12, 18, 19, 21, 22, 25, 26, 29, 30, 33 and 34. Measured values are given in Table 18 see "any other information on results" . pH was measured on Days 0, 4, 5, 11, 12, 18, 19, 25, 26, 29 and 34. Measured values are given in Table 19 "any other information on results"
Total hardness (mg/L CaCO3) was measured at the beginning of the test (on Day 0) in each test solutions (including control) and at the end of the test (on Day 34) in all test aquariums (see "any other information on results").

Recording

Test conditions were measured with suitable instruments and documented in the raw data.

Reference substance (positive control):
not specified
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Details on results:
Environmental Conditions

Temperature: The temperature was maintained within a range of 26 ± 1.5 °C and was appropriately constant. The overall temperature range in the test chambers was 25.1 - 26.5 °C with a maximum difference of 1.4 °C. The measured water temperature range in a concurrent test chamber (monitored continuously with a min/max thermometer) was 25.1 - 26.5 °C with a maximum difference of 1.4 °C. Results are summarized in Table 4 see "any other information on results". Measured temperature values are given in Table 16 and Table 17 see "any other information on results".

Oxygen concentration: Values over 60 % of the air saturation value (ASV) were observed during the test. The overall range in the test chambers was 60.7 % – 95.4 % ASV. Test solutions were slightly aerated from Day 5 to Day 24 (during the period when dry food was provided). Results are summarized in Table 5. Measured values are given in Table 18 see "any other information on results".

pH: Summarized results of the measured pH values are given Table 6 see "any other information on results". Measured values are given in Table 19 see "any other information on results".

Water Hardness: Water hardness was measured at the beginning and end of the test. Measured values are given in Table 7 see "any other information on results".

Analytical Results

1-ethoxy-2-(2-methoxyethoxy)ethane concentration in the test solutions was determined at 9 renewal periods. Five different concentrations and the control group were tested. The nominal test concentrations were 0.36 mg/L, 0.82 mg/L, 1.9 mg/L, 4.3 mg/L and 10 mg/L. Four replicates were tested per each test group. Samples were taken and analysed by GC-MS method. The analytical part of the study (method, determination, results) is attached see "any other information on results". The results of analytical determination of the test concentrations are given in Table 8 see "any other information on results". .

Biological Results

Since the analytically measured test item concentrations were within the range of ± 20 % of the nominal values over the test period of 34 days biological results are based on the nominal concentrations.
Survival, hatching, body weight and body length data in the test groups were compared to the control.

Hatching and Survival

Hatching of larvae started on Day 3 in each test chambers. All survival larvae were hatched in the control and also in the test groups on Day 4 (end of hatching period). Observations on hatching and the numbers of larvae hatched daily are given in Table 20 see "any other information on results". .
Test organisms were observed at least once a day during the test and numbers of living animals, the mortalities and numbers of animals with observed abnormalities (body deformities and/or abnormal behaviour) were also recorded. The observations are given in Table 21 see "any other information on results".

Data evaluation on hatching and survival was performed: hatching success, post-hatch success, overall survival and cumulative mortality were calculated. Tabulated results are given in Table 9 see "any other information on results".

Hatching success (%) varied between 72.0 % and 96.0 % in the test chambers. The mean values were within the range of 80.0 % and 91.0 %.
Post-hatch success in the test chambers varied between 70.8 % and 89.5 %. The mean values were within the range of 75.9 % and 80.2 %.

The following endpoints were statistically evaluated in the test groups in comparison with the control data:
- hatching on Day 3 and Day 4 (see Table 10 "any other information on results". )
- post-hatch survival for period Day 4 – 14 (feeding with dry food only; see Table 11 "any other information on results". )
- post-hatch survival for period Day 4 – 25 (feeding both with dry food and live food; see Table 12 "any other information on results". );
- post-hatch survival for the whole juvenile stage (Day 4 - Day 34, see Table 13 "any other information on results". ).

No statistically significant effect on the hatching was observed on Day 3 or on Day 4 at any test concentrations.
No statistical significance was observed for post-hatch survival during any stage of the test at any concentration.
Based on this it is considered that the test item did not influence the survival or hatchability of the test organism.

Body Weights and Body Lengths

At the end of the test all surviving fish were weighed. Total body weights were measured on a replicate basis due to small size of the fish (see Table 22 "any other information on results". ). Mean body weights calculated for the surviving individual fish were statistically evaluated. No statistically significant differences were observed (see Table 14 "any other information on results". ). No dose-related changes were observed between the test item treated groups.

Body length was measured for each individual fish (see Table 23 see "any other information on results". ). Mean body length per surviving individuals were statistically evaluated (see Table 15 "any other information on results". ). No statistically significant differences were observed. No dose-related changes were observed between the test item treated groups.

Symptoms of intoxication were recorded during the test with limited extent. Numbers of abnormal animals observed during the test are given in Table 21 see "any other information on results".
Although data were not statistically evaluated, no obvious treatment-related symptoms were considered. The observed abnormalities were mainly gross body deformities. The nature and frequency of the observed abnormalities in the test item treated groups did not differ from the control and did not show any dose dependence. Neither of the animals with observed abnormalities survived the test, since no fish were observed with abnormal appearance or behaviour at the end of the test.
Based on the overall results the following biological endpoints can be determined for 1-ethoxy-2-(2-methoxyethoxy)ethane.
Results with reference substance (positive control):
not specified in the study report.
Reported statistics and error estimates:
The statistical analysis was performed by ToxRat Professional (Version 3.3.0) software.
To determine NOEC/LOEC values for hatchability and post-hatch survival, Chi2 test with Bonferroni Correction was performed.
To determine NOEC/LOEC values for body weight and body length, Dunnett’s Multiple t-test (at the 0.05 significance level) was performed.
See "illustration".

Analytical Investigations: 


Measurement of Test Item Concentration in the Test Solutions


Table 1. Other materials

























Chemical



Grade



Manufacturer/ Supplier



Batch / Lot Number



Expiry/Retest date



Acetonitrile



for HPLC  super gradient 



VWR



19L021843



December 02, 2022



Ultra-pure water



ASTM Type I



prepared by Synergy smart UV HF, MILLIPORE



 


Table 2. Results of the method validation (Study No.: 103-100-6044)


































Specificity



Interference was less than 20 % of the lowest concentration of the calibration curve



Repeatability/Instrument precision  (7 injections)



1.8% (0.1 µg/mL) and 0.2% (8 µg/mL)



Linear range 



0.1 – 8 µg/mL



Recovery from the dilution water of OECD 210 test



                       107%  (0.3 mg/L) 


                       96%     (100 mg/L)



Stability of the Stock Solution (5 ± 3 °C)



at least 2 days



Stability in the Autosampler



at least 32 hours



Stability in dilution water of OECD 210 test at 26 ± 1.5 °C



at least 7 days



 


Table 3. Parameters of the calibration equations 








































































Analytical occasion



Constant



X Coefficient



R. Squared



November 12, 2021



-264



93673



1.000



November 16, 2021



313



92970



1.000



November 19, 2021



-171



94494



1.000



November 23, 2021



-313



75429



1.000



November 26, 2021



197



74479



0.999



November 30, 2021



-169



72770



1.000



December 03, 2021



197



73590



1.000



December 07, 2021



608



82128



0.999



December 13, 2021



-449



82606



1.000



December 16, 2021



37



76878



1.000



 


Table 4: Concentrations measured at the renewal period of November 12-16, 2021 




















































Nominal concentration mg/L



Start 1



End 1



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.374



104



0.342



95



0.82



0.833



102



0.825



101



1.9



1.96



103



1.90



100



4.3



4.46



104



4.43



103



10 



10.0



100



10.3



103



 


Table 5: Concentrations measured at the renewal period of November 16-19, 2021 




















































Nominal concentration mg/L



Start 2



End 2



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.360



100



0.387



108



0.82



0.849



103



0.886



108



1.9



1.97



104



2.06



108



4.3



4.60



107



4.69



109



10 



10.8



108



11.0



110



 


Table 6: Concentrations measured at the renewal period of November 19-23, 2021 




















































Nominal concentration mg/L



Start 3



End 3



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.368



102



0.398



111



0.82



0.834



102



0.868



106



1.9



1.43



75



1.45



76



4.3



4.46



104



4.58



106



10 



10.3



103



10.5



105



Table 7: Concentrations measured at the renewal period of November 23-26, 2021 




















































Nominal concentration mg/L



Start 4



End 4



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.359



100



0.377



105



0.82



0.894



109



0.961



117



1.9



1.86



98



2.00



105



4.3



4.23



98



4.57



106



10 



9.73



97



10.9



109



Table 8: Concentrations measured at the renewal period of November 26-30, 2021 




















































Nominal concentration mg/L



Start 5



End 5



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.350



97



0.417



116



0.82



0.812



99



0.909



111



1.9



1.92



101



2.08



109



4.3



4.34



101



4.87



113



10 



10.1



101



11.5



115



Table 9: Concentrations measured at the renewal period of November 30 – December 03, 2021




















































Nominal concentration mg/L



Start 6



End 6



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.390



108



0.405



112



0.82



0.880



107



0.910



111



1.9



2.05



108



2.15



113



4.3



4.59



107



4.76



111



10 



10.9



109



11.3



113



Table 10: Concentrations measured at the renewal period of December 03 - 07, 2021




















































Nominal concentration mg/L



Start 7



End 7



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.374



104



0.351



97



0.82



0.843



103



0.803



98



1.9



1.97



104



1.92



101



4.3



4.50



105



4.38



102



10 



10.4



104



10.2



102



 


 


 


Table 11: Concentrations measured at the renewal period of December 07 - 13, 2021




















































Nominal concentration mg/L



Start 8



End 8



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.315



88



0.376



104



0.82



0.741



90



0.820



100



1.9



1.77



93



1.86



98



4.3



4.05



94



4.36



101



10 



9.49



95



10.1



101



 


 


 


Table 12: Concentrations measured at the renewal period of December 13 - 16, 2021




















































Nominal concentration mg/L



Start 9



End 9



Concentration, mean (mg/L)  



% of the nominal



Concentration, mean (mg/L)  



% of the nominal



0.36



0.317



88



0.368



102



0.82



0.702



86



0.825



101



1.9



1.63



86



1.92



101



4.3



3.71



86



4.36



101



10 



8.80



88



10.8



108



 


Table 3. Preparation of the Stock and Test Solutions






















































































Date



Relative Day



Test item weighed


(g)



Final volume in the dilution water (mL)



Nominal concentration of the stock solution (mg/L)



Volume of the test solutions prepared


from the stock solution (mL)



2021.11.12.



0



0.0102



100



100



500



2021.11.16.



4



0.2007



200



1000



10000



2021.11.19.



7



0.2011



200



1000



10000



2021.11.23.



11



0.2001



200



1000



10000



2021.11.26.



14



0.2001



200



1000



10000



2021.11.30.



18



0.2043



200



1000



10000



2021.12.03.



21



0.2000



200



1000



10000



2021.12.07.



25



0.2003



200



1000



10000



2021.12.13.



31



0.2007



200



1000



10000



Table 2. Components of the dilution (reconstituted, ISO) water



































Substance



Nominal concentration in the reconstituted water


(mg/L)



Nominal concentration in the dilution water


(reconstituted water: purified water =  3:2)


(mg/L)



CaCl2 x 2 H2O


Batch No.: PP/2020/11623


Supplier: lach:ner


Retest date: 31 March 2023



294



176.4



MgSO4 x 7H2O


Batch No.: PP/2020/01386


Supplier: lach:ner


Retest date: 30 June 2024



123



73.8



NaHCO3


Lot No.: 19I024103


Supplier: VWR


Expiry date: 28 May 2024



65



39



KCl


Batch No.: K51320237


Supplier: Merck


Expiry date: 30 April 2024



5.8



3.5



Deionized water


 



prepared with BWT Christ Ministil ion exchanger resin systems (Serial No.: 935455) in the laboratory of TOXI-COOP Zrt.



Table 4 Temperature Ranges in the Test Chambers












































































































































































Nominal concentrations


(mg/L)



Parallels



 



Range (°C)



 



Minimum



Maximum



Difference



Control



1



25.2



26.5



1.3



 



2



25.2



26.5



1.3



 



3



25.2



26.5



1.3



 



4



25.2



26.4



1.2



0.36



1



25.2



26.5



1.3



 



2



25.2



26.3



1.1



 



3



25.2



26.4



1.2



 



4



25.2



26.2



1.0



0.82



1



25.1



26.2



1.1



 



2



25.1



26.2



1.1



 



3



25.1



26.1



1.0



 



4



25.2



26.3



1.1



1.9



1



25.1



26.2



1.1



 



2



25.1



26.4



1.3



 



3



25.2



26.3



1.1



 



4



25.1



26.3



1.2



4.3



1



25.1



26.2



1.1



 



2



25.2



26.3



1.1



 



3



25.2



26.3



1.1



 



4



25.3



26.3



1.0



10



1



25.1



26.2



1.1



 



2



25.2



26.4



1.2



Table 5 Dissolved Oxygen Concentration Ranges in the Test Chambers


























































































































































































Nominal concentrations


(mg/L)



Parallels



 



Range (% ASV)



 



Minimum



Maximum



Difference



Control



1



62.9



93.4



30.5



 



2



63.4



92.5



29.1



 



3



63.2



93.4



30.2



 



4



68.0



93.2



25.2



0.36



1



65.8



92.1



26.3



 



2



63.9



92.1



28.2



 



3



66.7



94.3



27.6



 



4



62.4



93.6



31.2



0.82



1



61.4



93.1



31.7



 



2



60.8



93.7



32.9



 



3



61.2



93.6



32.4



 



4



60.7



93.4



32.7



1.9



1



63.4



92.0



28.6



 



2



62.8



93.2



30.4



 



3



65.7



92.3



26.6



 



4



64.4



93.1



28.7



4.3



1



61.7



94.3



32.6



 



2



61.3



93.8



32.5



 



3



60.9



92.0



31.1



 



4



62.4



92.1



29.7



10



1



62.1



93.7



31.6



 



2



63.0



95.4



32.4



 



3



64.6



94.2



29.6



 



4



62.7



94.1



31.4



ASV = Air Saturation Value


 


Table 6 pH Ranges in the Test Chambers


























































































































































































Nominal concentrations


(mg/L)



Parallels



 



Range



 



Minimum



Maximum



Difference



Control



1



7.06



7.81



0.75



 



2



7.09



7.81



0.72



 



3



7.10



7.90



0.80



 



4



7.12



7.90



0.78



0.36



1



7.14



7.90



0.76



 



2



7.08



7.90



0.82



 



3



7.11



7.90



0.79



 



4



7.08



7.90



0.82



0.82



1



7.09



7.84



0.75



 



2



7.06



7.83



0.77



 



3



7.07



7.81



0.74



 



4



7.11



7.81



0.70



1.9



1



7.04



7.73



0.69



 



2



7.07



7.73



0.66



 



3



7.08



7.74



0.66



 



4



7.10



7.73



0.63



4.3



1



7.03



7.83



0.80



 



2



7.10



7.83



0.73



 



3



7.12



7.83



0.71



 



4



7.09



7.83



0.74



10



1



7.06



7.82



0.76



 



2



7.05



7.82



0.77



 



3



7.08



7.82



0.74



 



4



7.12



7.82



0.70



 


Table 7 Total Hardness (mg/L CaCO3) of the Test Solutions




























































































































Nominal concentrations


(mg/L)



Parallels



 



Days



0a



34b



Control


 



1


2



169.1



169.1


169.1



 



3



 



178.0



 



4



 



178.0



0.36


 



1


2



160.2



178.0


178.0



 



3



 



178.0



 



4



 



178.0



0.82


 



1


2



169.1



178.0


178.0



 



3



 



178.0



 



4



 



178.0



1.9


 



1


2



160.2



178.0


169.1



 



3



 



178.0



 



4



 



178.0



4.3


 



1


2



160.2



178.0


178.0



 



3



 



178.0



 



4



 



178.0



10


 



1


2



160.2



178.0 178.0



 



3



 



178.0



 



4



 



178.0



a = measured in the test solutions used for treatment of the eggs b = measured in the test chambers at the end of the test


 


Table 8      Summarized Results of Analytical Determination of the Test Concentrations (Determined for 9 Renewal Periods)



























































Nominal


Concentrations (mg/L)



Start of the Renewal Periods



End of the Renewal Periods



Mean measured


Concentrations (mg/L) with CI (95 %)



Percentage of the Nominal (%)



Mean Measured


Concentrations (mg/L) with CI (95 %)



Percentage of the Nominal (%)



Control



not detected



-



not detected



-



0.36



0.356 ± 0.020



99



0.380 ± 0.019



106



0.82



0.821 ± 0.048



100



0.867 ± 0.041



106



1.9



1.840 ± 0.153



97



1.927 ± 0.156



101



4.3



4.327 ± 0.223



101



4.556 ± 0.144



106



10



10.058 ± 0.505



101



10.733 ± 0.379



107



CI = Confidence Interval


Table 9 Evaluation of Data on Survival and Hatching




















































































































































































































































































































































































































































































































Nominal concentrations (mg/L)



 



Control



 



 



0.36



 



 



0.82



 



 



1.9



 



 



4.3



 



 



10



 



Parallels



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



Number of eggs treated



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



Total no. of larvae hatched (Day 4)



19



20



22



20



22



20



20



18



21



21



20



20



23



24



23



21



20



22



19



19



19



24



21



24



No. of fish at the end



15



15



19



15



17



17



15



15



17



16



15



17



18



17



18



16



15



16



17



15



15



19



17



18



Cumulative mortality (Day 0-34)



10



10



6



10



8



8



10



10



8



9



10



8



7



8



7



9



10



9



8



10



10



6



8



7



Sum:



36



 



 



 



36



 



 



 



35



 



 



 



31



 



 



 



37



 



 



 



31



 



 



 



Mortality Day 0-4


Sum:



6 19



5


 



3


 



5


 



3



5



5



7



4 18



4


 



5


 



5


 



2



1



2



4



5 20



3


 



6


 



6


 



6



1



4



1



20



 



 



 



9



 



 



 



12



 



 



 



Mortality Day 5-34



4



5



3



5



5



3



5



3



4



5



5



3



5



7



5



5



5



6



2



4



4



5



4



6



Sum:



17



 



 



 



16



 



 



 



17



 



 



 



22



 



 



 



17



 



 



 



19



 



 



 



Hatching success (%)



76.0



80.0



88.0



80.0



88.0



80.0



80.0



72.0



84.0



84.0



80.0



80.0



92.0



96.0



92.0



84.0



80.0



88.0



76.0



76.0



76.0



96.0



84.0



96.0



Mean: 



81.0



 



 



 



80.0



 



 



 



82.0



 



 



 



91.0



 



 



 



80.0



 



 



 



88.0



 



 



 



Post-hatch success (%)



78.9



75.0



86.4



75.0



77.3



85.0



75.0



83.3



81.0



76.2



75.0



85.0



78.3



70.8



78.3



76.2



75.0



72.7



89.5



78.9



78.9



79.2



81.0



75.0



Mean: 



78.8



 



 



 



80.2



 



 



 



79.3



 



 



 



75.9



 



 



 



79.0



 



 



 



78.5



 



 



 



Overall survival (%)



60.0



60.0



76.0



60.0



68.0



68.0



60.0



60.0



68.0



64.0



60.0



68.0



72.0



68.0



72.0



64.0



60.0



64.0



68.0



60.0



60.0



76.0



68.0



72.0



Mean: 



64.0



 



 



 



64.0



 



 



 



65.0



 



 



 



69.0



 



 



 



63.0



 



 



 



69.0



 



 



 



Cumulative mortality (%)



40.0



40.0



24.0



40.0



32.0



32.0



40.0



40.0



32.0



36.0



40.0



32.0



28.0



32.0



28.0



36.0



40.0



36.0



32.0



40.0



40.0



24.0



32.0



28.0



Mean: 



36.0



 



 



 



36.0



 



 



 



35.0



 



 



 



31.0



 



 



 



37.0



 



 



 



31.0



 



 



 



 


Table 10 Statistical Analysis of Data on Hatching






























































Nominal concentrations


(mg/L)



Number of eggs treated



Day 3#


Hatched



 


Nonhatched



Day 4$


Hatched



 


Nonhatched



Control



100



36



64



81



19



0.36



100



62 NS



38



80 NS



20



0.82



100



48NS



52



82 NS



18



1.9



100



44 NS



56



91 NS



9



4.3



100



39 NS



61



80 NS



20



10



100



40 NS



60



88 NS



12



Data were statistically evaluated by Chi2 2x2 Table Test with Bonferroni Correction (compared to the control). # = Start of hatching $ = End of hatching


NS = Not significant


 


Table 11 Statistical Analysis of Post-hatch Survival from Day 4 to Day 14









































Nominal concentrations


(mg/L)



Number of larvae survived


(on Day 4)



Number of larvae survived


(on Day 14)



Control



81



66



0.36



80



71 NS



0.82



82



70 NS



1.9



91



72 NS



4.3



80



69 NS



10



88



76 NS



Data were statistically evaluated by Chi2 2x2 Table Test with Bonferroni Correction (compared to the control).


NS = not significant


 


Table 12 Statistical Analysis of Post-hatch Survival from Day 4 to Day 25









































Nominal concentrations


(mg/L)



Number of larvae survived


(on Day 4)



Number of larvae survived


(on Day 25)



Control



81



64



0.36



80



65 NS



0.82



82



65 NS



1.9



91



69 NS



4.3



80



63 NS



10



88



69 NS



Data were statistically evaluated by Chi2 2x2 Table Test with Bonferroni Correction (compared to the control). NS = not significant


 


Table 13 Statistical Analysis of Post-hatch Survival from Day 4 to Day 34









































Nominal concentrations


(mg/L)



Number of larvae survived


(on Day 4)



Number of larvae survived


(on Day 34)



Control



81



64



0.36



80



64 NS



0.82



82



65 NS



1.9



91



69 NS



4.3



80



63 NS



10



88



69 NS



Data were statistically evaluated by Chi2 2x2 Table Test with Bonferroni Correction (compared to the control). NS = not significant


 


Table 14 Statistical Analysis of the Body Weight Data































































                     



 



 



Nominal concentrations (mg/L)



 



 



Parallels



Control



0.36



0.82



1.9



4.3



10


 

1


2 3 4



0.0175 0.0237


0.0213


0.0254



0.0219


0.0194


0.0217


0.0256



0.0218


0.0206


0.0205


0.0222



0.0213


0.0226


0.0219


0.0231



0.0195


0.0207


0.0237


0.0227



0.0221


0.0260


0.0191


0.0206



 



Mean



0.0220



0.0221 NS



0.0213 NS



0.0222 NS



0.0216 NS



0.0219 NS



 



SD



0.003



0.003



0.001



0.001



0.002



0.003



 



CV%



15.6



11.6



4.0



3.5



8.8



13.5



 


Data were statistically evaluated by Dunett’s Multiple t-test (α = 0.050; one-sided smaller)


               NS = not significant                       SD = Standard deviation                   CV = Coefficient of variation


 


Table 15 Statistical Analysis of the Body Length Data































































                     



 



 



Nominal concentrations (mg/L)



 



 



Parallels



Control



0.36



0.82



1.9



4.3



10


 

1


2 3


4



15,1


15.6


15.1


15.3



14.9


14.4


15.1


15.3



15.3


14.9 15.0


14.8



14.3


15.1 14.9


15.5



15.2


15.1 15.1


15.2



15.8 14.9


15.1


14.4



 



Mean



15.28



14.93 NS



15.00 NS



14.95 NS



15.15 NS



15.05 NS



 



SD



0.24



0.39



0.22



0.50



0.06



0.58



 



CV%



1.5



2.6



1.4



3.3



0.4



3.9



 


Data were statistically evaluated by Dunett’s Multiple t-test (α = 0.050; one-sided smaller)


               NS = not significant                       SD = Standard deviation                   CV = Coefficient of variation


 


Table 16      Measured Temperature Values (°C) 



















































































































































































































































































































































































































Nominal concentrations


[mg/L]



Parallels



 



 



 



 



 



 



 



Days/Measurements



 



 



 



 



 



 



 



0 T



1


1



2


1



3


1



4


1



2



1



5


2



6


1



2



7


1



2



8


1



2



9


1



2



Control



1


2



25.9


25.8



26.3


26.0



26.2


26.2



26.4


26.2



26.5


26.5



25.3 25.3



25.8


25.7



25.5 25.7



25.4


25.2



25.5 25.4



25.8 25.7



25.8


25.6



25.8 25.8



25.8


25.8



26.2 26.0



25.5


25.4



 



3



25.7



26.2



26.0



26.0



26.5



25.4



25.8



25.6



25.3



25.6



25.6



25.5



25.8



25.7



26.2



25.8



 



4



25.5



26.1



26.1



25.9



26.4



25.6



25.5



25.7



25.3



26.0



25.6



25.5



25.5



25.6



26.1



25.7



0.36



1


2



25.7


25.4



26.3


26.3



26.0


26.2



26.3


26.2



26.5


26.0



25.3


25.6



25.6


25.9



26.0


26.0



25.6


25.3



25.7


25.7



25.4


25.6



25.6


25.3



25.7


25.9



25.6


25.5



26.0


25.8



25.8


25.6



 



3



25.2



26.2



26.0



26.2



26.2



25.3



25.6



25.9



25.7



25.7



25.5



25.4



25.6



25.4



25.9



25.5



 



4



25.7



26.0



26.1



26.0



25.8



25.4



25.8



25.8



25.4



25.7



25.5



25.3



25.5



25.6



25.8



25.6



0.82



1


2



25.6


25.7



25.9


25.6



25.9


25.9



26.1


26.0



26.2


26.2



25.8


25.6



25.8


25.7



25.7


25.7



25.8


25.6



25.6


25.8



25.5


25.8



25.6


25.6



25.8


25.4



25.7


25.4



25.6


25.5



25.5


25.4



 



3



25.7



25.6



25.7



26.1



26.1



25.6



25.6



26.0



25.6



25.8



25.6



25.6



25.8



25.7



25.8



25.6



 



4



25.3



25.7



26.0



25.9



26.1



25.5



26.0



25.8



25.7



25.6



25.6



25.4



25.7



25.6



25.4



25.8



1.9



1


2



25.2


25.2



26.1


26.0



26.0


26.1



26.1


25.9



26.2


25.6



25.9


25.8



26.1


26.2



26.1


26.2



25.6


25.8



25.7


25.8



25.9


25.7



25.7


25.9



25.8


26.0



25.8


25.7



25.5


25.7



25.6


25.4



 



3



25.4



26.0



26.0



25.8



26.1



25.7



26.2



26.1



26.1



25.7



25.9



25.9



26.1



25.9



25.7



25.2



 



4



25.6



25.9



25.8



25.8



26.0



25.7



26.3



26.2



25.9



25.7



25.8



26.2



25.8



25.4



25.8



25.3



4.3



1


2



25.8


25.8



25.7


25.7



25.8


25.9



26.1


25.8



26.2


26.0



26.1


26.0



26.2


26.0



26.1


26.0



25.8


25.7



25.4


25.5



25.9


25.7



26.0


26.1



25.8


26.1



25.9


25.5



25.9


25.7



25.3


25.6



 



3



25.4



25.6



25.9



25.9



26.1



25.9



26.2



26.0



26.1



25.7



25.8



25.7



26.1



25.9



25.9



25.8



 



4



25.6



25.9



25.8



25.8



25.6



25.8



26.1



25.9



25.8



25.6



25.8



25.8



26.1



25.4



25.8



25.6



10



1


2



25.4


25.7



26.0


26.0



26.2


26.1



26.1


26.4



26.1


26.0



26.1


26.0



26.1


26.0



26.0


26.0



25.7


25.9



25.4


25.5



25.6


25.8



25.8


26.1



25.9


26.1



25.9


25.7



25.9


25.9



25.4


25.5



 



3



25.3



25.9



25.9



25.8



25.8



26.0



26.1



25.9



26.1



25.6



26.0



25.7



26.0



25.6



25.9



25.6



 



4



25.5



26.1



25.8



26.0



26.3



25.8



26.2



26.0



25.8



25.6



26.0



25.6



26.1



26.1



25.8



25.6



T = at the treatment



  • = before renewal of the test solution

  • = after renewal of the test solution



















































































































































































































































































































































































































Nominal concentrations


[mg/L]



Parallels



 



 



 



 



 



 



 



Days/Measurements



 



 



 



 



 



 



 



10


1



2



11


1



2



12


1



2



1



13 2



14


1



2



15


1



2



16


1



2



17


1



2



Control



1


2



26.1


25.6



25.6


25.4



26.1


25.8



25.6 25.7



25.4


25.3



25.3


25.4



26.2


26.0



25.8


25.7



25.3 25.5



25.2 25.8



25.5


25.4



25.7


25.9



26.0


25.7



25.7


25.7



25.8 25.4



25.7


25.6



 



3



25.9



25.5



26.0



25.9



25.3



25.6



26.0



25.8



25.6



25.8



25.4



26.1



25.6



25.4



25.7



25.4



 



4



25.6



25.5



25.8



25.7



25.3



25.3



26.1



25.7



25.6



26.1



25.6



25.8



25.6



25.5



25.8



25.4



0.36



1


2



25.7


25.9



25.6


25.5



25.8


25.8



25.6


25.4



25.3


25.2



25.3


25.3



25.8


25.8



26.1


25.7



25.8


25.6



25.7


25.4



25.8


25.3



25.9


25.4



25.5


25.3



25.2


25.2



25.4


25.2



25.2


25.3



 



3



25.8



25.6



25.8



25.3



25.2



25.3



25.9



25.8



25.4



25.9



25.2



25.6



25.2



25.2



25.3



25.3



 



4



25.7



25.6



25.7



25.5



25.3



25.4



25.8



25.8



25.4



25.6



25.3



25.8



25.5



25.3



25.3



25.6



0.82



1


2



25.7


25.5



25.6


25.4



25.5


26.1



25.3


25.2



25.3


25.4



25.3


25.2



25.6


25.5



25.4


25.3



25.6


25.8



25.4


25.3



25.2


25.4



25.7


25.8



25.2


25.4



25.3


25.1



25.4


25.2



25.3


25.4



 



3



25.6



25.4



25.7



25.3



25.4



25.3



25.8



25.4



25.8



25.4



25.5



25.6



25.2



25.2



25.2



25.6



 



4



25.8



25.9



25.8



25.6



25.6



25.6



25.5



25.5



26.0



25.3



25.6



25.6



25.7



25.2



25.6



25.6



1.9



1


2



26.0


26.0



25.7


25.8



26.1


26.4



25.8


25.8



26.0


26.1



26.1


25.9



25.5


25.7



25.7


26.1



26.1


26.4



25.7


25.6



25.7


26.1



25.6


25.4



25.8


25.8



25.4


25.7



25.4


25.6



25.3


25.6



 



3



25.9



25.8



26.3



25.8



26.1



25.7



25.7



25.7



26.2



25.6



25.8



25.4



26.0



25.6



25.7



25.8



 



4



26.0



25.6



26.3



26.1



26.2



25.8



26.0



26.1



26.3



25.3



25.8



25.5



26.1



25.6



26.0



25.7



4.3



1


2



26.0


25.8



25.9


26.1



26.0


26.2



25.8


26.0



25.8


26.1



25.6


25.7



25.9


25.7



25.2


25.4



26.1


26.2



25.3


25.4



26.2


26.3



25.8


25.7



26.2


26.2



25.6


25.8



26.0


26.1



25.3


25.7



 



3



26.1



26.0



26.2



25.8



26.2



25.7



25.9



25.8



26.3



25.3



26.1



25.4



26.3



25.7



25.6



25.6



 



4



25.9



25.8



26.2



26.2



26.1



26.0



25.8



26.1



26.3



25.8



26.1



25.4



26.2



25.8



26.1



25.8



10



1


2



25.7


25.8



25.5


25.5



26.0


26.1



25.8


26.0



25.8


26.1



25.6


26.1



25.9


25.9



25.4


25.9



26.1


26.2



25.8


25.7



26.1


26.3



25.3


25.7



25.9


25.9



25.9


25.9



26.0


26.1



25.3


25.7



 



3



25.8



25.6



26.2



25.8



26.3



26.0



25.9



25.8



26.3



25.7



26.3



25.7



25.9



25.7



25.6



25.6



 



4



26.1



25.6



26.3



26.2



26.1



26.0



26.0



26.1



26.4



25.4



26.3



25.7



26.2



25.8



26.1



25.8




  • = before renewal of the test solution

  • = after renewal of the test solution



















































































































































































































































































































































































































Nominal concentrations


[mg/L]



Parallels



 



 



 



 



 



 



 



Days/Measurements



 



 



 



 



 



 



 



18


1



2



19


1



2



20


1



2



1



21 2



22


1



2



23


1



2



24


1



2



25


1



2



Control



1


2



26.0


26.0



25.5


25.5



25.4


25.3



26.0


25.3



25.7


25.3



25.6 25.8



26.1


25.7



25.7


26.0



26.3


26.1



25.2 25.2



26.2


25.9



25.7


26.0



26.1 25.7



25.9


25.9



26.1 25.4



25.4


25.2



 



3



26.1



25.5



25.4



25.8



25.3



25.4



25.6



25.9



26.3



25.2



26.1



25.3



26.1



25.7



25.4



26.0



 



4



25.9



25.3



25.2



25.7



25.8



25.8



26.1



26.2



25.4



25.3



26.4



25.8



26.2



25.9



25.8



25.3



0.36



1


2



25.3


25.2



25.9


25.3



25.5


25.4



25.8


25.9



25.7


26.0



25.2


25.2



25.9


26.2



26.1


26.1



25.4


25.8



25.2


25.2



26.4


26.3



25.7


25.8



26.2


26.0



26.0


26.0



25.6


25.8



25.8


25.7



 



3



25.3



25.5



25.4



25.4



25.3



25.2



25.7



25.7



25.6



25.3



26.4



25.9



26.2



26.1



25.4



25.8



 



4



25.2



25.6



25.6



25.4



25.8



25.4



26.1



25.9



25.8



25.2



26.2



25.4



26.0



25.9



25.8



25.9



0.82



1


2



25.4


25.4



25.2


25.3



25.3


25.5



25.8


25.6



25.7


25.8



25.3


25.2



25.4


25.4



25.3


26.0



25.4


25.8



25.3


25.4



25.8


26.1



26.2


26.1



25.3


25.8



25.7


25.9



25.6


25.8



25.4


25.5



 



3



25.6



25.4



25.6



25.8



25.9



25.1



25.8



25.3



25.6



25.2



26.0



25.9



25.7



25.9



25.9



25.6



 



4



25.6



25.8



26.0



25.4



25.4



25.2



25.6



25.8



25.8



25.3



25.8



26.3



25.8



26.0



26.3



26.0



1.9



1


2



25.3


25.2



25.6


25.9



26.1


26.1



25.8


25.6



25.1


25.1



25.1


25.2



25.8


25.4



25.7


25.8



25.5


25.7



25.3


25.2



25.4


25.6



26.2


26.1



25.9


25.4



25.8


25.4



26.2


26.1



25.4


25.6



 



3



25.3



25.7



26.0



25.8



25.2



25.3



25.8



25.9



25.6



25.3



25.6



25.8



25.6



25.4



25.7



25.6



 



4



25.2



25.9



26.2



25.1



25.3



25.4



25.6



26.3



25.7



25.2



25.7



26.0



25.8



25.8



25.8



25.7



4.3



1


2



26.1


26.0



26.0


26.1



26.2


26.1



25.1


25.2



25.1


25.6



25.4


25.8



25.8


25.8



26.2


26.1



25.8


26.0



26.0


25.3



25.7


26.1



25.4


25.4



25.7


26.1



25.6


25.8



25.9


26.3



25.7


26.1



 



3



25.9



25.8



26.2



25.2



25.5



25.6



25.7



25.9



26.1



25.8



26.1



25.6



26.0



25.4



26.2



25.8



 



4



25.8



26.0



25.9



25.3



25.3



25.8



25.8



26.3



26.1



25.7



26.1



25.7



26.1



25.8



26.1



25.7



10



1


2



25.3


25.4



26.0


26.1



26.2


26.1



25.1


25.2



25.1


25.6



25.4


25.8



25.6


25.8



26.2


26.1



25.8


26.0



25.8


25.9



25.7


26.1



25.4


25.4



25.7


26.1



25.6


25.8



25.9


26.3



25.8


25.6



 



3



25.2



25.8



26.0



25.2



25.5



25.6



25.7



25.5



26.1



25.4



26.1



25.6



26.0



25.8



26.2



25.8



 



4



25.5



26.0



26.2



25.3



25.3



25.8



25.8



25.9



26.1



25.5



26.1



25.7



26.1



26.0



26.1



26.2




  • = before renewal of the test solution

  • = after renewal of the test solution


























































































































































































































































































Nominal concentrations


[mg/L]



Parallels



 



 



 



 



Days/Measurements



 



 



 



26


1



27


1



28


1



29


1



30


1



31


1



2



32


1



33


1



34


1



Control



1


2



25.6


25.6



25.7


25.3



25.2


25.3



25.7


25.4



25.6


25.4



25.6


25.3



25.6


25.4



26.0


25.8



26.2


26.0



26.0 25.9



 



3



25.7



25.6



25.6



25.6



25.6



25.6



25.7



25.8



26.1



26.0



 



4



25.3



25.8



25.3



25.8



25.2



25.9



25.7



25.9



26.3



26.1



0.36



1


2



25.5


25.2



25.8


26.1



25.6


25.2



25.9


26.0



25.3


25.3



25.9


25.8



25.6


25.6



26.1


26.2



25.6


25.2



26.2


26.2



 



3



25.3



25.7



25.2



25.2



25.3



25.6



25.2



25.8



25.6



26.0



 



4



25.6



25.8



26.1



25.3



25.4



25.7



25.3



26.0



25.3



26.1



0.82



1


2



25.3


25.6



25.6


25.7



26.2


25.8



25.6


25.3



25.5


25.8



25.2


25.2



25.1


25.6



25.4


25.7



26.0


25.9



25.7


25.8



 



3



25.7



25.3



26.0



25.6



25.8



25.4



25.4



25.4



25.6



25.6



 



4



25.4



25.5



25.4



25.7



25.6



25.5



25.7



25.4



25.7



25.7



1.9



1


2



25.8


26.0



25.2


25.3



25.7


25.4



25.6


25.3



25.6


26.1



25.7


25.3



25.8


25.9



25.4


25.3



25.5


25.5



25.3


25.4



 



3



25.3



25.6



25.2



26.1



26.1



25.8



25.7



25.3



25.7



25.6



 



4



25.3



25.3



25.2



26.2



25.6



25.8



26.0



25.3



25.4



25.4



4.3



1


2



25.4


25.8



25.6


25.7



25.2


25.3



25.8


26.0



25.3


25.6



25.6


25.2



25.1


25.7



25.4


25.5



25.7


25.6



25.6


25.6



 



3



25.8



25.6



25.6



25.4



25.3



25.6



25.5



25.8



26.2



26.1



 



4



25.9



25.9



25.7



25.7



25.6



25.3



25.7



25.8



25.9



26.1



10



1


2



25.7


25.7



25.6


25.7



25.2


25.3



25.4


25.3



25.7


25.6



26.0


26.1



25.4


25.2



25.4


25.5



25.7


25.6



25.6


25.6



 



3



25.5



25.6



25.6



25.2



25.7



25.9



25.3



25.7



26.2



26.1



 



4



25.3



25.9



25.7



25.4



25.6



25.8



25.2



25.6



25.9



26.1



1 = before renewal of the test solution


 


Table 17 Measured Minimum-Maximum Temperatures (°C) in the Concurrent Test Chamber






































































































































































 



 



 



 



 



 



 



 



Days



 



 



 



 



 



 



 



0



1



2



3



4



5



6



7



8



9



10



11



12



13



14



Minimum



-



25.3



25.4



25.2



25.2



25.2



25.1



25.2



25.2



25.1



25.3



25.2



25.1



25.2



25.1



Maximum



-



26.4



26.4



26.4



26.2



26.5



26.5



26.5



26.4



26.4



26.4



26.4



26.2



26.2



26.5



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Days



 



 



 



 



 



 



 



15



16



17



18



19



20



21



22



23



24



25



26



27



28



29



Minimum



25.3



25.2



25.1



25.2



25.1



25.3



25.1



25.2



25.4



25.5



25.2



25.1



25.2



25.1



25.1



Maximum



26.5



26.3



26.3



26.1



26.2



26.4



26.3



26.5



26.4



26.4



26.3



26.5



26.4



26.4



26.3



 





































 



 



 



Days



 



 



30



31



32



33



34



Minimum



25.1



25.2



25.1



25.2



25.3



Maximum



26.4



26.0



26.3



26.0



26.3



Table 18      Measured Dissolved Oxygen Concentrations (% ASV)






























3              90.2        89.5        92.0        63.2        89.9        71.4



89.8



73.5



91.5



73.0



93.4



71.8



92.5



72.1        90.5        72.1        93.0        75.4        73.0        66.7        70.0



71.0



4                                                                                              73.2



92.1



70.5



92.0



70.9



93.0



75.0



92.9



 



 























































































































































































 



75.0



89.5



73.3



91.0



69.8



92.1



74.2



91.4



91.4



74.7



90.4



76.2



90.7



71.0



91.1



74.0



92.1



92.4



72.6



92.9



69.9



89.9



74.6



89.7



74.6



94.3



 



71.4



92.0



73.2



90.0



72.0



92.1



71.2



90.8



 



70.9



89.5



69.7



90.5



72.5



93.0



71.0



90.6



92.7



72.1



89.9



72.0



92.0



72.8



92.5



71.9



92.7



90.9



73.0



91.8



72.4



92.1



70.1



90.9



72.8



93.6



 



74.1



92.0



73.4



90.0



73.2



90.4



75.0



93.4



 



70.6



89.2



70.4



89.9



69.9



90.4



70.4



90.7



93.2



71.8



90.3



69.9



91.9



70.1



91.6



74.3



90.2



91.7



73.8



91.2



71.0



90.8



72.3



92.3



73.9



90.5



 



73.1



91.3



73.5



89.8



69.8



93.1



73.1



92.3



 



75.4



92.1



72.1



90.2



71.0



94.3



76.0



90.9



93.8



70.9



92.0



71.1



91.6



72.1



93.8



74.1



91.1



91.6



71.3



91.6



73.3



91.2



70.8



90.1



73.3



91.3



 



72.6



90.8



69.8



90.0



69.9



90.1



72.4



90.8























0.36



1


2


3


4



0.82



1


2


3


4



1.9



1


2


3


4



4.3



1


2


3


4



                                              91.3        89.7        90.3                                                                                    63.9 71.2                                                              89.9        75.1        92.0                                                                                        77.3                                                              71.9        68.1        70.2                                                                                        69.2


                                              91.3        89.7        92.9                                                                                    66.7 70.0                                                              90.4        74.2        90.8                                                                                        76.2                                                              72.2        67.0        70.1                                                                                        70.4


                                              89.9        88.9        91.8                                                                                    60.8 69.9                                                              93.7        71.8        91.3                                                                                        75.8                                                              70.2        69.7        70.3                                                                                        70.9


                                              89.9        88.9        92.0                                                                                    61.2 72.5                                                              91.1        73.0        92.0                                                                                        77.0                                                              71.0        67.8        71.0                                                                                        72.4


                                              92.0        88.4        89.9                                                                                    62.8 71.6                                                              93.1        72.5        90.5                                                                                        75.8                                                              71.1        71.3        70.2                                                                                        70.1


                                              92.0        88.4        91.0                                                                                    65.7 72.2                                                              90.2        72.2        90.0                                                                                        74.3                                                              69.9        70.0        70.3                                                                                        69.8


                                              91.1        87.2        92.3                                                                                    61.3 72.5                                                              90.1        75.8        91.6                                                                                        73.6                                                              72.0        71.2        68.7                                                                                        69.6


                                              91.1        87.2        91.4                                                                                    60.9 71.4                                                              92.0        73.0        89.9                                                                                        74.9                                                              70.5        70.0        70.2                                                                                        70.4



  • 0            89.9            72.8            90.2            74.0            90.4            75.0    93.1           

  • 688.1            89.7            63.0            95.4            74.1            89.7            71.9            90.4            73.1            89.8    75.4            92.5            74.1            89.9            74.2            93.0            76.1            70.6            69.4            68.9            71.2


10



  • 688.1            90.5            64.6            94.2            73.1            91.2            70.0            89.9            71.0            90.0    73.0            92.7            74.2            91.5            74.8            91.8            74.9            69.8            69.9            71.5            69.9

  • 688.1            89.9            62.7            94.1            70.8            90.4            69.9            93.1            71.6            91.6    72.9            90.6            71.6            90.6            72.6            91.5            74.6            71.1            71.0            70.9            68.9



  • = = measured in the prepared test solutions used for treatments a* = measured in the old test solution pooled according to treatment group (at transfer of the animals) a = measured in the clean solution after the transfer b = measured in the old solution before the transfer


Table 19 Measured pH values




































































































































































































































































Nominal concentrations


[mg/L]



Parallels



 



 



 



 



 



 



Days



 



 



 



 



 



0


*



4 a*



4 a



5 b



11 a



12 b



18 a



19 b



25 a



26


 



29


 



34


 



Control



1


2



7.81


7.81



7.79


7.79



7.76


7.81



7.20


7.18



7.74


7.80



7.35


7.30



7.65


7.79



7.18


7.11



7.72


7.81



7.06


7.20



7.21


7.09



7.25


7.18



 



3



7.81



7.79



7.82



7.19



7.90



7.41



7.89



7.10



7.76



7.20



7.19



7.13



 



4



7.81



7.79



7.88



7.22



7.82



7.38



7.90



7.23



7.85



7.22



7.13



7.12



0.36



1


2



7.90


7.90



7.83


7.83



7.85


7.76



7.19


7.30



7.89


7.85



7.38


7.32



7.90


7.84



7.16


7.15



7.85


7.80



7.39


7.08



7.15


7.13



7.14


7.11



 



3



7.90



7.83



7.77



7.29



7.76



7.33



7.85



7.15



7.79



7.24



7.15



7.11



 



4



7.90



7.83



7.73



7.25



7.81



7.25



7.90



7.24



7.76



7.24



7.08



7.13



0.82



1


2


3



7.81


7.81


7.81



7.81


7.81


7.81



7.74


7.71


7.67



7.20


7.44


7.35



7.78


7.72


7.70



7.27


7.26


7.31



7.78


7.83


7.75



7.21


7.19


7.10



7.84


7.81


7.76



7.28


7.16


7.07



7.19


7.06


7.11



7.09


7.11


7.20



 



4



7.81



7.81



7.60



7.41



7.69



7.38



7.76



7.11



7.69



7.17



7.12



7.16



1.9



1


2


3



7.70


7.70


7.70



7.73


7.73


7.73



7.69


7.68


7.74



7.23


7.41


7.25



7.68


7.72


7.70



7.22


7.22


7.21



7.73


7.68


7.71



7.10


7.10


7.08



7.65


7.65


7.71



7.04


7.24


7.16



7.10


7.12


7.13



7.09


7.07


7.18



 



4



7.70



7.73



7.71



7.21



7.73



7.29



7.71



7.15



7.70



7.12



7.20



7.10



4.3



1


2


3



7.83


7.83


7.83



7.76


7.76


7.76



7.72


7.71


7.71



7.32


7.33


7.25



7.69


7.72


7.73



7.19


7.23


7.31



7.75


7.71


7.71



7.09


7.15


7.12



7.73 7.83


7.74



7.28 7.31


7.36



7.03 7.19


7.12



7.12 7.10


7.13



 



4



7.83



7.76



7.68



7.29



7.73



7.32



7.76



7.12



7.69



7.09



7.12



7.09



10



1


2


3



7.82


7.82


7.82



7.68


7.68


7.68



7.70


7.65


7.69



7.22


7.23


7.23



7.68


7.70


7.75



7.23


7.25


7.28



7.72


7.69


7.72



7.18


7.05


7.18



7.71 7.66


7.66



7.10 7.21


7.25



7.06 7.19


7.08



7.09 7.08


7.09



 



4



7.82



7.68



7.62



7.40



7.69



7.32



7.72



7.12



7.68



7.21



7.16



7.12




  • = = measured in the prepared test solutions used for treatments a* = measured in the old test solution pooled according to treatment group (at transfer of the animals) a = measured in the clean solution after the transfer b = measured in the old solution before the transfer


Table 20 Number of Larvae Hatched and Duration of Hatching











































































































































































































Nominal concentrations


[mg/L]



Parallels



Number of eggs treated



 



 



Days



 



 



0



1



2



3



4



Control



1


2



25


25



0


0



0


0



0


0



10


8



19


20



 



3



25



0



0



0



12



22



 



4



25



0



0



0



6



20



0.36



1


2



25


25



0


0



0


0



0


0



19


18



22


20



 



3



25



0



0



0



16



20



 



4



25



0



0



0



9



18



0.82



1


2



25


25



0


0



0


0



0


0



11


14



21


21



 



3



25



0



0



0



7



20



 



4



25



0



0



0



16



20



1.9



1


2



25


25



0


0



0


0



0


0



8 11



23


24



 



3



25



0



0



0



18



23



 



4



25



0



0



0



7



21



4.3



1


2



25


25



0


0



0


0



0


0



3 16



20


22



 



3



25



0



0



0



8



19



 



4



25



0



0



0



12



19



10



1


2



25


25



0


0



0


0



0


0



12


4



19


24



 



3



25



0



0



0



11



21



 



4



25



0



0



0



13



24



 


Table 21 Number of Living and Dead Embryo/Larvae/Fish  and Number of Abnormalities Observed During the Test
























































































































































































































































































































































































































































































































































































































































































































































































































































































Nominal Concentrations (mg/L)



 



 



 



Control



 



 



 



 



 



 



0.36



 



 



Parallels



 



1



 



2



 



 



3



 



 



4



 



1



 



2



 



3



 



 



4



Days



S



 



M



S



M



S



 



M



S



 



M



S



M



S



M



S



M



S



M



0



25



 



0



25



0



25



 



0



25



 



0



25



 



0



25



0



25



0



25



0



1



19



 



6



20



5



22



 



3



20



 



5



22



 



3



20



5



20



5



19



6



2



19



 



0



20



0



22



 



0



20



 



0



22



 



0



20



0



20



0



18



1



3



19



 



0



20



0



22



 



0



20



 



0



22



 



0



20



0



20



0



18



0



4



19



 



0



20



0



22



 



0



20



 



0



22



 



0



20



0



20 (2)



0



18



0



5



19



 



0



20



0



22



 



0



20



 



0



22



 



0



20



0



20 (2)



0



18



0



6



19



 



0



19



1



22



 



0



20



 



0



21



1



20



0



20 (2)



0



18



0



7



18 (1)



 



1



19



0



21



 



1



18



 



2



21



 



0



20



0



20 (2)



0



17



1



8



18 (1)



 



0



18



1



21



 



0



18



 



0



21



0



20



0



20 (2)



0



17



0



9



18 (1)



 



0



18



0



21



 



0



18



 



0



20



 



1



20



0



20 (2)



0



16



1



10



18 (1)



 



0



18



0



21



 



0



18



 



0



20



0



20



0



18



2



16



0



11



17



 



1



17



1



20



 



1



17



 



1



20



 



0



20



0



18



0



16



0



12



17



 



0



17



0



20



 



0



16



 



1



20



0



20



0



18



0



16



0



13



16



 



1



17



0



20



 



0



16



 



0



20



 



0



20



0



18



0



16



0



14



16



 



0



16



1



19



 



1



15



 



1



19



1



19



1



17



1



16



0



15



16



 



0



16



0



19



 



0



15



 



0



19 (1)



 



0



19 (1)



0



17 (1)



0



16



0



16



16



 



0



16 (1)



0



19



 



0



15



 



0



19 (1)



0



19 (1)



0



17 (1)



0



16



0



17



16



 



0



16 (1)



0



19



 



0



15



 



0



19 (1)



 



0



19 (1)



0



17 (1)



0



16



0



18



15



 



1



16 (1)



0



19



 



0



15



 



0



19 (1)



0



19 (1)



0



17 (1)



0



16



0



19



15



 



0



16 (1)



0



19



 



0



15



 



0



19 (1)



 



0



19 (1)



0



17 (1)



0



16



0



20



15



 



0



16 (1)



0



19



 



0



15



 



0



18



1



18



1



16



1



16



0



21



15



 



0



16 (1)



0



19



 



0



15



 



0



18



 



0



18



0



16



0



16



0



22



15



 



0



15



1



19



 



0



15



 



0



18



0



18



0



15



1



16



0



23



15



 



0



15



0



19



 



0



15



 



0



18



 



0



18



0



15



0



15



1



24



15



 



0



15



0



19



 



0



15



 



0



17



1



18



0



15



0



15



0



25



15



 



0



15



0



19



 



0



15



 



0



17



 



0



18



0



15



0



15



0



26



15



 



0



15



0



19



 



0



15



 



0



17



0



18



0



15



0



15



0



27



15



 



0



15



0



19



 



0



15



 



0



17



 



0



18



0



15



0



15



0



28



15



 



0



15



0



19



 



0



15



 



0



17



0



18



0



15



0



15



0



29



15



 



0



15



0



19



 



0



15



 



0



17



 



0



18



0



15



0



15



0



30



15



 



0



15



0



19



 



0



15



 



0



17



0



17



1



15



0



15



0



31



15



 



0



15



0



19



 



0



15



 



0



17



 



0



17



0



15



0



15



0



32



15



 



0



15



0



19



 



0



15



 



0



17



0



17



0



15



0



15



0



33



15



 



0



15



0



19



 



0



15



 



0



17



 



0



17



0



15



0



15



0



34



15



 



0



15



0



19



 



0



15



 



0



17



0



17



0



15



0



15



0



Table 21 (continued) Number of Living and Dead Embryo/Larvae/Fish and Number of Abnormalities Observed During the Test


















































































































































































































































































































































































































































































































































































































































































































































































































































Nominal Concentrations (mg/L)



 



 



 



0.82



 



 



 



 



 



1.9



 



 



Parallels



 



1



 



2



 



 



3



 



4



 



1



 



2



 



3



 



 



4



Days



S



 



M



S



M



S



 



M



S



M



S



M



S



M



S



M



S



M



0



25



 



0



25



0



25



 



0



25



0



25



 



0



25



0



25



0



25



0



1



23



 



2



21



4



20



 



5



20



5



23



 



2



24



1



24



1



23



2



2



23 (1)



 



0



21 (1)



0



20



 



0



20



0



23



 



0



24 (1)



0



24



0



23 (1)



0



3



22



 



1



21 (1)



0



20



 



0



20 (1)



0



23



 



0



24 (1)



0



24



0



21



2



4



21 (2)



 



1



21 (1)



0



20 (1)



 



0



20 (1)



0



23 (3)



 



0



24 (3)



0



23 (2)



1



21 (2)



0



5



21 (2)



 



0



21 (1)



0



20 (1)



 



0



20 (1)



0



22 (2)



 



1



23 (2)



1



22 (1)



1



20 (1)



1



6



21 (2)



 



0



21 (1)



0



19



 



1



20 (1)



0



21 (2)



1



21



2



21



1



20 (1)



0



7



20 (1)



 



1



20



1



19



 



0



19



1



19



 



2



20



1



21



0



18



2



8



20



 



0



20



0



19



 



0



19



0



19



0



20



0



20



1



18



0



9



18



 



2



19



1



19



 



0



18



1



19



 



0



20



0



20



0



18



0



10



18



 



0



18



1



17



 



2



18



0



19



0



19



1



20



0



18



0



11



18



 



0



18



0



17



 



0



18



0



19



 



0



19



0



20



0



18



0



12



18



 



0



18



0



17



 



0



18



0



19



0



19



0



20



0



18



0



13



18



 



0



18



0



17



 



0



18



0



19



 



0



19



0



20



0



18



0



14



18



 



0



18



0



17



 



0



17



1



18



1



18



1



19



1



17



1



15



18



 



0



18



0



17



 



0



17



0



18



 



0



18



0



19



0



17



0



16



18



 



0



17



1



16



 



1



17



0



18



0



18



0



18



1



17



0



17



18



 



0



17



0



16



 



0



17



0



18



 



0



18



0



18



0



17



0



18



18



 



0



17



0



16



 



0



17



0



18



0



17



1



18



0



17



0



19



18



 



0



17



0



16



 



0



17



0



18



 



0



17



0



18



0



17



0



20



17



 



1



17



0



16



 



0



17



0



18



0



17



0



18



0



16



1



21



17



 



0



17



0



15



 



1



17



0



18



 



0



17



0



18



0



16



0



22



17



 



0



17



0



15



 



0



17



0



18



0



17



0



18



0



16



0



23



17



 



0



16



1



15



 



0



17



0



18



 



0



17



0



18



0



16



0



24



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



25



17



 



0



16



0



15



 



0



17



0



18



 



0



17



0



18



0



16



0



26



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



27



17



 



0



16



0



15



 



0



17



0



18



 



0



17



0



18



0



16



0



28



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



29



17



 



0



16



0



15



 



0



17



0



18



 



0



17



0



18



0



16



0



30



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



31



17



 



0



16



0



15



 



0



17



0



18



 



0



17



0



18



0



16



0



32



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



33



17



 



0



16



0



15



 



0



17



0



18



 



0



17



0



18



0



16



0



34



17



 



0



16



0



15



 



0



17



0



18



0



17



0



18



0



16



0



Table 21 (continued) Number of Living and Dead Embryo/Larvae/Fish and Number of Abnormalities Observed During the Test
























































































































































































































































































































































































































































































































































































































































































































































































































































































Nominal Concentrations (mg/L)



 



 



 



4.3



 



 



 



 



 



 



10



 



 



Parallels



 



1



 



2



 



 



 



3



 



4



 



1



 



2



 



3



 



 



4



Days



S



 



M



S



M



 



S



 



M



S



M



S



M



S



M



S



M



S



M



0



25



 



0



25



0



 



25



 



0



25



0



25



 



0



25



0



25



0



25



0



1



20



 



5



23



2



 



20



 



5



19



6



20



 



5



24



1



22



3



24



1



2



20



 



0



23 (1)



0



 



20



 



0



19 (2)



0



20



 



0



24 (1)



0



22 (1)



0



24



0



3



20



 



0



22



1



 



19



 



1



19



0



20



 



0



24 (1)



0



21



1



24



0



4



20 (1)



 



0



22



0



 



19



 



0



19 (1)



0



19 (1)



 



1



24



0



21



0



24



0



5



19



 



1



22



0



 



19



 



0



18



1



19 (1)



 



0



23



1



21



0



24



0



6



19



 



0



20



2



 



18



 



1



18



0



18



1



22



1



21



0



22



2



7



19



 



0



19



1



 



18



 



0



18



0



17



 



1



22



0



19



2



22



0



8



19



 



0



19



0



 



18



 



0



18



0



17



0



22



0



19



0



22



0



9



19



 



0



19



0



 



18



 



0



18



0



17



 



0



22



0



19



0



22



0



10



18



 



1



19



0



 



18



 



0



18



0



17



0



22



0



19



0



22



0



11



18



 



0



19



0



 



18



 



0



18



0



17



 



0



22



0



19



0



22



0



12



18



 



0



19



0



 



18



 



0



18



0



17



0



21



1



19



0



22



0



13



18



 



0



19



0



 



18



 



0



18



0



17



 



0



21



0



19



0



22



0



14



17



 



1



18



1



 



17



 



1



17



1



17



0



20



1



18



1



21



1



15



17



 



0



18



0



 



17



 



0



17



0



17



 



0



20



0



18



0



21



0



16



17



 



0



18



0



 



17



 



0



17



0



17



0



19



1



18



0



20



1



17



17 (1)



 



0



18 (1)



0



 



17



 



0



17



0



16



 



1



19



0



17



1



19



1



18



16 (1)



 



1



18 (1)



0



 



17



 



0



17



0



16



0



19



0



17



0



19



0



19



16 (1)



 



0



18 (1)



0



 



17



 



0



17



0



16 (1)



 



0



19



0



17



0



19



0



20



15



 



1



17



1



 



17



 



0



17



0



16 (1)



0



19



0



17



0



19



0



21



15



 



0



17



0



 



17



 



0



17



0



15



 



1



19



0



17



0



18



1



22



15



 



0



16



1



 



17



 



0



17



0



15



0



19



0



17



0



18



0



23



15



 



0



16



0



 



17



 



0



17



0



15



 



0



19



0



17



0



18



0



24



15



 



0



16



0



 



17



 



0



16



1



15



0



19



0



17



0



18



0



25



15



 



0



16



0



 



17



 



0



15



1



15



 



0



19



0



17



0



18



0



26



15



 



0



16



0



 



17



 



0



15



0



15



0



19



0



17



0



18



0



27



15



 



0



16



0



 



17



 



0



15



0



15



 



0



19



0



17



0



18



0



28



15



 



0



16



0



 



17



 



0



15



0



15



0



19



0



17



0



18



0



29



15



 



0



16



0



 



17



 



0



15



0



15



 



0



19



0



17



0



18



0



30



15



 



0



16



0



 



17



 



0



15



0



15



0



19



0



17



0



18



0



31



15



 



0



16



0



 



17



 



0



15



0



15



 



0



19



0



17



0



18



0



32



15



 



0



16



0



 



17



 



0



15



0



15



0



19



0



17



0



18



0



33



15



 



0



16



0



 



17



 



0



15



0



15



 



0



19



0



17



0



18



0



34



15



 



0



16



0



 



17



 



0



15



0



15



0



19



0



17



0



18



0



 


Table 22 Body Weight Data






































































































































































































































Nominal concentrations



Parallels



Number of fish



Total body weighta



Mean


body weightb



(mg/L)



 



 



(g)



(g)



Control



1



15



0.262



0.0175



 



2



15



0.356



0.0237



 



3



19



0.404



0.0213



 



4



15



0.381



0.0254



 



Mean



-



0.351



0.0220



0.36



1



17



0.372



0.0219



 



2



17



0.330



0.0194



 



3



15



0.325



0.0217



 



4



15



0.384



0.0256



 



Mean



-



0.353



0.0221



0.82



1



17



0.371



0.0218



 



2



16



0.330



0.0206



 



3



15



0.308



0.0205



 



4



17



0.377



0.0222



 



Mean



-



0.347



0.0213



1.9



1



18



0.384



0.0213



 



2



17



0.384



0.0226



 



3



18



0.395



0.0219



 



4



16



0.370



0.0231



 



Mean



-



0.383



0.0222



4.3



1



15



0.292



0.0195



 



2



16



0.331



0.0207



 



3



17



0.403



0.0237



 



4



15



0.341



0.0227



 



Mean



-



0.342



0.0216



10



1



15



0.331



0.0221



 



2



19



0.494



0.0260



 



3



17



0.325



0.0191



 



4



18



0.371



0.0206



 



Mean



-



0.380



0.0219



a = Total body weights were measured on a replicate basis. b = Mean body weights were calculated for the surviving individual fish.


Table 23 Body Length Data


































































































































































































































































































































































Nominal


Concentrations (mg/L)



 



Control



 



 



0.36



 



 



0.82



 



 



1.9



 



 



 



 



4.3



 



 



 



10



 



Parallels



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4


 

1.75


1.80


1.80


1.60


1.50


1.65


1.65


1.45


1.50


1.30


1.45


1.25


1.25



1.70


1.75


1.45


1.55


1.75


1.50


1.60


1.60


1.40


1.75


1.55


1.75


1.25



1.45


1.60


1.50


1.60


1.35


1.80


1.55


1.45


1.55


1.55


1.05


1.50


1.55



1.50


1.60


1.85


1.60


1.65


1.60


1.60


1.85


1.60


1.60


1.55


1.25


1.10



1.60


1.50


1.55


1.60


1.45


1.55


1.55


1.60


1.60


1.65


1.45


1.50


1.30



1.45


1.50


1.60


1.40


1.45


1.40


1.40


1.50


1.45


1.55


1.45


1.35


1.35



1.60


1.45


1.50


1.45


1.50


1.50


1.55


1.40


1.35


1.45


1.85


1.65


1.45



1.35


1.60


1.65


1.60


1.60


1.60


1.60


1.55


1.60


1.70


1.55


1.55


1.45



1.50


1.45


1.45


1.50


1.60


1.65


1.60


1.55


1.50


1.55


1.50


1.55


1.60



1.35


1.75


1.65


1.35


1.60


1.45


1.45


1.55


1.50


1.60


1.25


1.45


1.35



1.20


1.55


1.40


1.35


1.20


1.45


1.40


1.80


1.55


1.65


1.60


1.25


1.85



1.65


1.35


1.50


1.55


1.40


1.45


1.60


1.60


1.45


1.85


1.60


1.20


1.35



1.20


1.65


1.45


1.30


1.20


1.40


1.45


1.55


1.65


1.60


1.60


1.55


1.30



1.70


1.60


1.75


1.50


1.75


1.65


1.60


1.45


1.65


1.55


1.35


1.50


1.45



1.65


1.35


1.85


1.60


1.45


1.35


1.50


1.75


1.50


1.35


1.45


1.35


1.35



1.65


1.50


1.70


1.45


1.60


1.50


1.60


1.60


1.60


1.80


1.35


1.50


1.50



1.10


1.60


1.55


1.50


1.60


1.55


1.50


1.70


1.75


1.60


1.25


1.45


1.40



1.65


1.45


1.35


1.35


1.75


1.60


1.55


1.55


1.40


1.25


1.80


1.30


1.40



1.60


1.65


1.50


1.50


1.60


1.55


1.20


1.65


1.60


1.45


1.45


1.45


1.50



1.45


1.45


1.50


1.55


1.40


1.45


1.45


1.65


1.60


1.55


1.55


1.60


1.60



1.70


1.65


1.60


1.75


1.45


1.50


1.70


1.30


1.50


1.50


1.50


1.75


1.70



1.25


1.60


1.45


1.50


1.60


1.30


1.45


1.45


1.45


1.55


1.50


1.35


1.60



1.60


1.50


1.45


1.25


1.45


1.55


1.50


1.60


1.50


1.40


1.45


1.55


1.70



1.70


1.55


1.45


1.60


1.50


1.60


1.45


1.45


1.40


1.30


1.30


1.15


1.60



 



1.35



1.45



1.60



1.45



1.50



1.40



1.45



1.05



1.65



1.50



1.65



1.45



1.30



1.20



1.45



1.55



1.65



1.55



1.75



1.60



1.65



1.55



1.75



1.40



 



1.30



1.30



1.55



1.20



1.25



1.25



1.50



1.50



1.45



1.45



1.60



1.35



1.50



1.35



1.35



1.50



1.60



1.55



1.50



1.40



1.45



1.50



1.65



1.35



 



 



 



1.60



 



1.30



1.45



 



 



1.35



1.60



 



1.50



1.40



1.45



1.50



1.35



 



1.65



1.30



 



 



1.35



1.40



1.45



 



 



 



1.45



 



1.35



1.50



 



 



1.50



 



 



1.35



1.35



1.25



1.35



 



 



 



1.35



 



 



1.35



1.40



1.45



 



 



 



1.45



 



 



 



 



 



 



 



 



 



1.30



 



1.70



 



 



 



 



 



 



1.65



 



1.20



 



 



 



1.45



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



1.80



 



 



Mean



1.51



1.56



1.51



1.53



1.49



1.44



1.51



1.53



1.53



1.49



1.50



1.48



1.43



1.51



1.49



1.55



1.52



1.51



1.51



1.52



1.58



1.49



1.51



1.44



SD



0.19



0.17



0.15



0.21



0.12



0.08



0.12



0.16



0.08



0.13



0.20



0.15



0.15



0.16



0.16



0.12



0.17



0.16



0.14



0.08



0.13



0.13



0.12



0.14



Group mean



1.52



 



 



 



1.49



 



 



 



1.50



 



 



 



1.49



 



 



 



1.51



 



 



 



1.50



 



 



 



Number of fish measured



15



15



19



15



17



17



15



15



17



16



15



17



18



17



18



16



15



16



17



15



15



19



17



18



 


Table 24 Feeding of Test Animals (Larvae/Juvenile)































































Days



0



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



DF mg/day/test aquarium



 



No food supplied



 



12.5



 



 



 



 



 



25



 



 



 



 



 



AN



 



 



 



 



N



o live food supplied



 



 



 



 



 



 



 



Not specified amount*



 



 















































































Days



18



19



20



21



22



23



24



25



26



27



28



29



30



31



32



33



34



DF mg/day/test aquarium



 



25



 



 



12.5



 



 



 



 



No dry food



 



 



 



 



No food supplied



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



AN



 



 



 



 



 



 



Not specified amount*



 



 



 



 



 



 



 



 



DF = Dry food (Sera micron Nature)


AN = Artemia naupli


* Both DF and AN were supplied ad libitum (means supplying excessive amount of food at any time of feeding). No amount of artemia nauplies were determined in the “suspension” supplied to each test chambers in same volume.


 


 

Validity criteria fulfilled:
yes
Remarks:
The water temperature met with the validity criteria. The dissolved oxygen concentration in the test solutions also met with the criteria. According to this the assay is considered valid and the obtained results are reliable.
Conclusions:
Under the conditions of this fish early life stage toxicity test, the obtained results showed that the test item 1-ethoxy-2-(2methoxyethoxy)ethane had no significant lethal or sub-lethal effects on early life stages of Zebrafish (Danio rerio) up to at least the highest tested concentration of 10 mg/L. Accordingly, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on Zebrafish (Danio rerio) up to at least the tested maximum concentration of 10 mg/L.
Executive summary:
























Test Item:



1-ethoxy-2-(2-methoxyethoxy)ethane



Test species:



Zebrafish (Danio rerio)



Purpose:



The purpose of this study was to evaluate the chronic toxicity of the test item 1-ethoxy-2-(2-methoxyethoxy)ethane to early life stages (embryo, larvae and juveniles) of zebrafish (Danio rerio). For this purpose, eggs were exposed in a semi static test to aqueous test media containing the test item for 34 days at a range of concentrations under defined conditions. Lethal and sub-lethal effects were assessed and compared with the solvent control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC).



Dilution water:



Reconstituted water (ISO medium) diluted with purified water (in a ratio of 3 : 2 = reconstituted water: purified water)



Control:



Negative control (dilution water)



Test Item Concentrations, Formulation:






























































 



 



Test concentrations were selected according to the results of the preliminary test (acute 96 hours fish test on Zebrafish) and in line of the OECD 210 guideline. The selected nominal concentrations (spaced by a factor of 2.3) were 0.36 mg/L, 0.82 mg/L, 1.9 mg/L, 4.3 mg/L and 10 mg/L which are the same as was used in the Daphnia magna 21-day reproduction test performed previously in the testing laboratory  (Study No.: 103-211-6041).



 



Analytics:



For determination of 1-ethoxy-2-(2-methoxyethoxy)ethane concentration samples were taken from the test solutions and from the negative control at the start and at the end of the renewal periods. Test item concentrations were analysed in the test solutions by a GC-MS method. 1-ethoxy-2-(2-methoxyethoxy)ethane concentration was determined at 9 renewal periods.



 



Test design:



One hundred eggs (attributed randomly to the groups and distributed equally between the four replicates) were tested per test concentrations and the control. Based on microscopic observation embryos were at approximately 2 to 32 cell stages at start of the treatment (in the  pre-treatment solutions). Test solutions were renewed in each test chamber either with replenishment of the test solutions (in the mornings) or with transfer of the test animals into a clean solution (in the afternoons).


Test organisms were kept in the incubator until Day 4 [until the test animals in the test chambers (glass Petri-dishes) become free-feeding]. Larvae were transferred to the test chambers (glass aquariums) on Day 4 before elimination of yolk sac and feeding was initiated.


 Animals were fed from the free-feeding stage to the end of the test with appropriate type and amount of food ad libitum. The same amount of food was supplied for each test chamber twice a day (except on Day 4) in excess at the time of renewal of the test solutions. 



 



Food was added to the test chambers either after replenishment of the test solutions or after transfer of the test animals into a clean test chambers.


Test organisms were observed once a day (mainly at the time of the transfers). At the end of the test animals were euthanized with hypothermic shock.



 



Measurements:



Environmental conditions (temperature, dissolved oxygen concentration, pH, water hardness) were monitored during the test.



 



Observations:



Mortality, hatching, body weight, body length, abnormal appearance/behaviour were observed during the test.



 



Statistics:


Results:



The statistical analysis was performed by ToxRat Professional (Version 3.3.0) software. 


To determine NOEC/LOEC values for hatchability and post-hatch survival, Chi2 test with Bonferroni Correction was performed. 


To determine NOEC/LOEC values for body weight and body length, Dunnett’s Multiple t-test (at the 0.05 significance level) was performed.



 



Validity:



The water temperature met with the validity criteria and the test item was analytically determined at regular intervals. Hatching success as well as post-hatch success in the control group were over the specified value of 70 % or 75 % (respectively).


The dissolved oxygen concentration in the test solutions also met with the criteria (the measured values were over the 60 % of the air saturation value during the test).



 



 



According to this the assay is considered valid and the obtained results are reliable.



 



Analytical results:



The analytically measured test item concentrations (determined at 9



 


    

renewal periods) were within the range of ± 20 % of the nominal values over the test period of 34 days. 


Biological results: Based on the results of the concentration analysis biological results were reported according to the nominal test concentrations which were 0.36 mg/L, 0.82 mg/L, 1.9 mg/L, 4.3 mg/L and 10 mg/L.


Hatching, post-hatch survival, body weight and body length data were evaluated compared to the control.


Number of larvae hatched daily was statistically evaluated on Day 3 and Day 4. No statistical significance was observed for hatching at any test concentrations.


Post-hatch survival was statistically evaluated on Days 14, 25 and 34. No statistical significance was observed for hatching at any test concentrations on any of the days.


 Total body weights were measured on a replicate basis. Mean body weights (calculated from the total body weights for the individual fish) were statistically evaluated. No statistically significant differences were observed at any test concentrations. 


                               Body length was measured for each individual fish and the data were


statistically evaluated. No statistically significant differences were observed. 


 Other sub-lethal effects were also monitored and numbers of embryos/larvae/juveniles with deformities and / or abnormal behaviour were recorded when observed. Although these data were not statistically evaluated, no obvious test item related changes in the appearance or behaviour of the animals were observed.


 


               Table 1.    Summary of Biological Endpoints






















































Endpoints



1-ethoxy-2-(2-methoxyethoxy)ethane concentration


(mg/L)



Hatchability NOEC



≥ 10



Hatchability LOEC



> 10



Post-hatch survival NOEC



≥ 10



Post-hatch survival LOEC



> 10



Body weight (terminal) NOEC



≥ 10



Body weight (terminal) LOEC



> 10



Body length (terminal) NOEC



≥ 10



Body length (terminal) LOEC



> 10



34 day NOEC



≥ 10



34 day LOEC



> 10



34 day ECx



> 10



 


Conclusion:             Under the conditions of this fish early life stage toxicity test, the obtained results showed that the test item 1-ethoxy-2-(2methoxyethoxy)ethane had no significant lethal or sub-lethal effects on early life stages of Zebrafish (Danio rerio) up to at least the highest tested concentration of 10 mg/L. Accordingly, the test item  1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on Zebrafish (Danio rerio) up to at least the tested maximum concentration of 10 mg/L.

Description of key information

Under the conditions of this fish early life stage toxicity test, the
obtained results showed that the test item 1-ethoxy-2-(2-
methoxyethoxy)ethane had no significant lethal or sub-lethal effects
on early life stages of Zebrafish (Danio rerio) up to at least the
highest tested concentration of 10 mg/L. Accordingly, the test item
1-ethoxy-2-(2-methoxyethoxy)ethane had no chronic effects on
Zebrafish (Danio rerio) up to at least the tested maximum
concentration of 10 mg/L.


 


Table 1. Summary of Biological Endpoints






















































Endpoints



1-ethoxy-2-(2-methoxyethoxy)ethane concentration


(mg/L)



Hatchability NOEC



≥ 10



Hatchability LOEC



> 10



Post-hatch survival NOEC



≥ 10



Post-hatch survival LOEC



> 10



Body weight (terminal) NOEC



≥ 10



Body weight (terminal) LOEC



> 10



Body length (terminal) NOEC



≥ 10



Body length (terminal) LOEC



> 10



34 day NOEC



≥ 10



34 day LOEC



> 10



34 day ECx



> 10



 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
>= 10 mg/L

Additional information