Reaction products of diphenylamine with nonene, branched

Long-term toxicity to fish

Administrative data

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of Benzeneamine, N-phenyl- with nonene (branched)

EC Number:

701-385-4

Cas Number:

36878-20-3

Molecular formula:

C21H29N - C30H47N (main constituents)

IUPAC Name:

Reaction products of Benzeneamine, N-phenyl- with nonene (branched)

Test material form:

liquid: viscous

Details on test material:

uvcb substance

Specific details on test material used for the study:

uvcb substance

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal loading rate: 10 mg/L, 0 mg/L
- Sampling method:
taken from alternating test replicates,
analytically verified twice during 7 days of exposure from freshly prepared test media on study day 0 (start of the exposure), 5, 8, 12, 15, 19, 22, 26, 29 and 33 and from corresponding 24 hours aged test media on study days 1, 6, 9, 13, 16, 20, 23, 27, 30, 34
Four aliquots were taken as samples after preparation of monitoring the limit loading rate without addition of solvent.
One replicate of the undiluted sample was centrifuged with 10000 g at room temperature for 5 minutes; another replicate was centrifuged with 4000 rpm at room temperature for 5 minutes, one replicate was measured without any pre-treatment. The remaining replicate was stored at ambient conditions.
Two additional samples were diluted with acetonitrile containing 0.2% formic acid (factor 2) directly after sampling.
The results indicated that centrifugation was not useful (due to adsorption effects).
No further pre-treatment was done and the replicates with addition of solvent were analyzed.

- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fractions (WAF)
appropriate volume of the test item was pipetted onto the surface of the dilution water (in 2 L flasks). For this purpose, the density was taken into account. From day 0 to day 20, 2 flasks per concentration were prepared, from day 21 to day 34, 8 flasks per concentration were prepared. A slow stirring procedure was applied for 72 ± 1 hour at the test temperature. Stirring regime was based on the results of a preliminary test. After a separation phase of 30 minutes at test temperature, the WAF was removed by siphoning (from the bottom of the glass flask). The resulting water accommodated fraction (WAF) was used for testing.
A coating phase (saturation of the test vessels) was carried out. The test vessels of the limit loading rate were pre-treated with the appropriate test media for at least 12 hours (over night) under test conditions. Before the start of the exposure and at the day of change of the test vessels (day 20), the test container were emptied and refilled with freshly prepared test solutions.
- Controls: treated as test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAF was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative for all test media.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: single brood stock at test facility (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 15 – 35 adult zebrafish were kept in at least 3 separate aquaria
- Method of collection of fertilised eggs: The fish were healthy with a mortality rate < 5% during the last 7 days before test initiation, and not medically treated during these 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After approximately 1 hour the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded. 250 eggs were taken and washed in dilution water. Eggs originated from 2 different spawnings. At least 70% of the eggs appeared healthy.
- Subsequent handling of eggs: Eggs that were used to start exposure were pooled and attributed randomized (eggs were placed in alternating groups into each of the three test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs)
- Fertilization check: Eggs with only a 2 cell stage were regarded as not fertilized and were discarded. Fertilization rate 86%

POST-HATCH FEEDING
- Start date: day 5, 2 days after the beginning of hatching (on study day 5 (post-hatch day 1).
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34)
- Type/source of feed: Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany)) and brine shrimp nauplii (48 h old) until the end of the test (ST-1: 2 – 4 times daily; brine shrimp: 2 – 7 times daily), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 5 times daily).
- Amount given: ad libitum

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

34 d

Remarks on exposure duration:

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

Test conditions

Hardness:

control: 57-62; mean 60 [mg CaCO3/L]
10 mg/L loading rate: 61-80; mean 65 [mg CaCO3/L]

Test temperature:

control: fresh medium (0d): 25.4 - 26.4, aged medium (1d): 25.0 - 26.2; mean 25.8°C
10 mg/L loading rate: fresh medium (0d): 25.2 - 26.4, aged medium (1d): 24.8 - 26.1; mean 25.8°C

pH:

control: fresh medium (0d): 7.10 - 7.78, aged medium (1d): 7.15 - 7.66; mean 7.4
10 mg/L loading rate: fresh medium (0d): 7.21 - 7.71, aged medium (1d): 7.41 - 7.70; mean 7.48

Dissolved oxygen:

control:min 62, max 99; mean 90%
10 mg/L loading rate: min 65, max 97; mean 90%

Conductivity:

143 µS/cm

Nominal and measured concentrations:

nominal loading rate: 10 mg/L, 0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Days 0 to 20: Crystallisation dishes were used. The volume of the test media in the dishes was about 700 mL. Days 20 to test end: Glass aquaria (22 x 22 x 7.3 cm) were used. The volume of the test media was about 3.5 L. Test vessels were covered with transparent lids. Juveniles were gently transferred to larger aquaria on study day 20.
- Aeration: The dilution water used for preparation of the test media was aerated. Study day 0 to 7: No aeration. Study day 8 to 34 (test end): Since measurements of the dissolved oxygen saturation on study day 8 were in the range of 62 to 83 %, a gentle aeration was applied for all test vessels.
- Renewal rate of test solution (frequency/flow rate): For the daily renewal of the test media the test organisms were retained in the test vessels whilst a proportion of 75 % of the media was changed. Old test media was gently removed by siphoning with glass tubes. Care was taken not to remove or injure the test organisms. Freshly prepared media was gently added with glass tubes to the test vessels to avoid stressing of the test organisms.
- Cleaning: The test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and
biodegradation of the test compound. Cleaning started on study day 5 (post hatch day 1).
- No. of fertilized eggs/embryos per vessel and replicates: 4 replicates per limit loading rate and control, with 20 eggs each (80 eggs per limit loading and control) For the whole study 160 eggs/fish were used.
- Biomass loading rate: A loading rate not exceeding 1 g/L wet weight fish was maintained.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same as used for holding. Tap water. Filtered on activated charcoal and aerated for at least 24h to remove chlorine
- Total organic carbon: 0.668 - 1.06, mean 0.828 mg/L determinations once per week on study days 1, 7, 13, 21, 33 and 34
- Chlorine: measured from the dilution water supply tank once per week on study days 0, 7, 14, 21, 28 and 34: < 0.01 mg/L.
- pH: 6.0-8.5
- Alkalinity: 0.6mmol/L (recent measuremnt April 2019)
- Acidity: 0.2 mmol/L (recent measurement: April 2019)
- Conductivity: 143 µS/cm (recent measurement: April 2019)
- Intervals of water quality measurement:
- Total hardness: once per week in one replicate of the control group and the limit loading rate;
- pH-value and temperature in all replicates of each test group from freshly prepared test media at the start of an exposure interval and same measurements from the corresponding interval of the aged test media
- dilution water temperature in control vessels: once per hour,
- dissolved oxygen: daily in all replicates of each test group from fresh and old test media;
- light intensity: start of exposure and on the day of changing the test vessels (study day 20)
- TOC and Chlorine from the dilution water

OTHER TEST CONDITIONS
- Photoperiod: 16 / 8 h photoperiod (light / dark)
- Light intensity: target: 300 ± 150 Lux. 168 to 370 lux (mean 294 lux) for the crystallization dishes (study day 0); 298 to 402 lux (mean 351 lux) for the test aquaria

EFFECT PARAMETERS MEASURED DAILY :
Hatching:
The number of hatched larvae until study day 5. Eggs were only removed, when mortality of eggs/embryos was observed as specified below.
On study day 4, 100 % of the control larvae had hatched. Therefore, study day 4 was defined as post-hatch day 0 (= PHD 0). For evaluation of hatch, every all hatched larvae (even dead ones) were counted. The cumulative number of hatched larvae was used for evaluation.

Mortality:
Criteria for mortality vary according to life stage:
- For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded.
- For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.

Further effects
Abnormal appearance and behavior were also recorded at adequate intervals. The number of larvae or fish showing abnormality of body form was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate.  

Measurement of fish size:
At the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was measured to the nearest 0.5 mm with graph paper. The total length (from the tip of the snout to the tip of the longer lobe of the caudal fin) was measured.

Measurement of fish wet weight:
At the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis to the nearest 0.1 mg. Fish were blotted on paper towels to remove excess moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.


POST-HATCH DETAILS
- Begin of post-hatch period: study day 4 with a hatching rate success of 100% in the control and 98% in the limit loading rate

FERTILIZATION SUCCESS STUDY
- 129 eggs were introduced in the control media and 18 of these eggs were discarded.
- 133 eggs were introduced in the limit loading rate and 18 of these eggs were discarded.
In total 36 eggs of 262 introduced eggs were discarded, resulting in a fertilization rate of 86%.

STATISTICAL EVALUATION:
For each parameter analyzed, survival (mortality), hatching success, wet weight and length data the following statistical tests were conducted for determination of the NOELR/LOELR:
The Fisher`s Exaxt Binomial test for hatching success at study day 4 and survival at study day 34 was done with a significance level of 0.05.
The Shapiro-Wilk’s test on normal distribution of fresh weight and length was done with a significance level of 0.01.
The Levene’s test on variance homogeneity of fresh weight and length was done with a significance level 0.01.
The Student-t test for comparison of fresh weight and length of treatments was done with significance level 0.05.
The ELRx -values and corresponding confidence intervals werederived from the results as far as possible. As the study was carried out as a limit test, no calculation was done.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

34 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate (WAF)

Basis for effect:

number hatched

Remarks on result:

other: hatchability

Remarks:

post hatch day 4: 98% eggs hatched at 10mg/L loading rate

Effect concentrations 2

Key result

Duration:

34 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate (WAF)

Basis for effect:

mortality

Remarks on result:

other: post hatch and overall mortality

Effect concentrations 3

Key result

Duration:

34 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate (WAF)

Basis for effect:

other: fry growth (length and weight)

Details on results:

No statistically significant differences were detected between the dilution water control and the limit loading rate of 10 mg/L of the test item. In conclusion under the conditions of this test the test item did not affect early-life stages of the zebrafish at a nominal loading rate of 10 mg/L of the water accommodated fraction.

- Egg fertilization rate: The egg fertilization rate, determined on study day 0 (start of the exposure) was 86 %. Eggs were fully covered with the respective test solutions during fertilization check. 129 eggs were introduced in the control media and 18 of these eggs were discarded. 133 eggs were introduced in the limit loading rate and 18 of these eggs were discarded. In total 36 eggs of 262 introduced eggs were discarded, result ing in a fertilization rate of 86%.
- Days to hatch or time to release of young: Hatching began on study day 3 in the control and the limit loading rate of the test item and continued until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate success of 100% in the control and 98% in the limit loading rate. Hatching was completed in the limit loading rate at study day 5 (100% hatching success)
Statistical procedures were applied for the total number of test organisms that have hatched. The Fisher`s Exact Binomial Test (alpha = 0.05) was performed for statistical analysis of hatching success on study day 4. No statistically significant difference was found between the control and the limit loading rate of 10 mg/L.
The NOELR and the LOELR (nominal limit loading rate) for this endpoint were determined to be 10 mg/L and > 10 mg/L, respectively.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): Swim-up was observed for a 2-day period from study days 3 to 5 . Newly hatched fry began to swim up on study day 4 (PHD 0). On study day 5 (PHD 1), all surviving larvae had swum up. No statistical analysis of swim-up data was carried out.
- Fry Survival (Post-Hatch Survival): The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75 %). The fry survival (post-hatch survival) at the end of the study was 79 % in the control. The post-hatch survival in the limit loading rate of the test item was 81 %
The Fisher`s Exact Binomial Test (alpha = 0.05) was performed for statistical analysis of post hatch survival data on study day 34 (PHD 30). No statistically significant differences were found for the nominal limit loading rate of 10 mg/L.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal limit loading rate), respectively.
The LLR0 value for post hatch survival on study day 34 (PHD 30) was determined to be 10 mg/L (nominal limit loading rate).
- Overall survival: The overall survival at the end of the exposure, related to the number of eggs introduced on day 0 was 79 % in the control group and 81 % in the limit loading rate of the test item .
The Fisher`s Exact Binomial Test (alpha = 0.05) was performed for statistical analysis of overall survival data on study day 34 (PHD 30). No statistically significant differences were found for the nominal limit loading rate of 10 mg/L.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal limit loading rate), respectively.
The LLR0 value for overall survival on study day 34 (PHD 30) was determined to be 10 mg/L (nominal limit loading rate
- Fry Growth
Fry growth, expressed as length and wet weight, was measured on study day 34 (PHD 30) from all survivors.
With the statistical procedures applied for fresh weight data and mean total length of all survivors (Student-t test for homogenous variances) the nominal limit loading rate of 10 mg/L showed no significant differences for these parameters. Therefore the NOELR and the LOELR for both parameters were are laid down as 10 mg/L and > 10 mg/L (nominal limit loading rate). For details refer to part 13.3 and 13.4
- Biomass Loading
The biomass-loading factor for the study was determined from the fresh weights of the fish control fish and the at fish of the limit loading rate at the end of the exposure.
The maximum biomass at the end of the exposure was determined in replicate 4 of the limit loading rate: 585.9 mg total fish weight. The maximum biomass loading based on the 3.5 liter volume of a single growth chamber was 167 mg/L.
This loading was well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
- Morphological and Behavioral Effects
No biologically significant morphological effects were observed in any treatment.
- Vital Eggs and Larvae from Study Days 0 to 34
from 20 eggs at day 0 per replicate, 20 larvae per replicate hatched .
- Dead Eggs and Larvae from Study Days 0 to 34
days with observed dead larvae: control: 11, 12, 13, 15, 18, 20, 23, 25, 31, 34 , Loadingrate 10mg/L: 12, 14, 15, 16, 18, 22, 23, 30, 31, 32, 33
dead larvae in replicates at day 34: control: 5, 3, 4, 5; Loadingrate 10mg/L: 1, 8, 5, 1

LC-MS/MS Analysis
The limit loading rate of the water accommodated fraction (WAF) of the test item and the control were analytically verified twice during 7 days of exposure from freshly prepared test media on study day 0 (start of the exposure), 5, 8, 12, 15, 19, 22, 26, 29 and 33 and from corresponding 24 hours aged test
media on study days 1, 6, 9, 13, 16, 20, 23, 27, 30 and 34 (end of the exposure). Based on the results of the pre-test, the analytics were confined to the
detection of the mono-alkylated Isomers.
The measured concentrations of the water accommodated fraction (WAF) of the test item in the fresh media were either extremely low (1.1 – 18.1 µg/L) or generally even below the limit of quantification (<1 μg/L).
In 75% fresh media and 25 % old media, the measured concentrations were up to 10.6 µg test item/L. In the old media, the measured concentrations were up to 3.78 µg test item/L.
It was concluded that these results could not be used to base test concentration upon. As a consequence, results of the present study were based on the loading rates initially prepared.
All reasonable efforts were taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD guidance document No. 23 (2019), for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures).

At the end of the exposure on day 20 and on day 34 the adsorption of the test item on the glass walls was investigated. The test item
did adsorb to the surface of the glass walls during exposure phase, but it is not possible to validate the analytical measurement of adsorbed test item (after desorption by use of a solvent). Therefore, the extent of desorption remains uncertain and gives only a qualitative estimate rather than an exact quantitative assessment of desorption.
 

Reported statistics and error estimates:

Hatching success day 4: Fisher`s Exact Binomial Test: p(exact) test concentration 1.000. There is no statistically significant difference between Control and WAF 10mg/L.

Post hatch and overall survival day 34:Fisher`s Exact Binomial Test: p(exact) test concentration 0.723. There is no statistically significant difference between Control and WAF 10mg/L.

Fry Growth: Mean Total Length on Study Day 34
Shapiro-Wilk´s Test on Normal Distribution: Shapiro-Wilk´s; p > 0.01
Levene´s Test on Variance Homogeneity (with Residuals): The Levene test indicates variance homogeneity (p > 0.010). Variance homogeneity check was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances: p(t) test concentration 0.997
There is no statistically significant difference between Control and WAF 10 mg/L.

Fry Growth: Fresh Weight on Study Day 34
Shapiro-Wilk´s Test on Normal Distribution: Shapiro-Wilk´s; p > 0.01
Levene´s Test on Variance Homogeneity (with Residuals): The Levene test indicates variance homogeneity (p > 0.010). Variance homogeneity check was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances: p(t) test concentration 0.988
There is no statistically significant difference between Control and WAF 10 mg/L.

Any other information on results incl. tables

Hatching Success in the Control and the Limit Loading Rate

Nominal test item loading rate [mg/L]	Rep.	PHD -2	PHD -1	PHD 0	PHD 1
		Study day 2	Study day 3	Study day 4	Study day 5
		Hatching success [%] per study day
Control	1	0	5	100	100
	2	0	15	100	100
	3	0	5	100	100
	4	0	25	100	100
	Mean	0	13	100	100
10	1	0	0	100	100
	2	0	0	100	100
	3	0	0	95	100
	4	0	5	95	100
	Mean	0	1	(-) 98	100

Percent Swim-up of Hatched Live Fry of the Control and the Limit Loading Rate

Nominal test item loading rate [mg/L]	Rep.	PHD -1	PHD 0	PHD 1
		Study day 3	Study day 4	Study day 5
		Swim up [%]
Control	1	0	25	100
	2	0	20	100
	3	0	35	100
	4	0	35	100
	Mean	0	29	100
10	1	0	70	100
	2	0	65	100
	3	0	90	100
	4	0	90	100
	Mean	0	79	100

Post-Hatch Survival on Study Day34 (PHD 30) of the Controland the Limit Loading Rate

Nominal test item loading rate [mg/L]	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae/post hatch survival on study day 34 (PHD 30) [%]
Control	1	20	17	85
	2	20	15	75
	3	20	16	80
	4	20	15	75
	Mean	20.0	15.8	79
10	1	20	19	95
	2	20	12	60
	3	20	15	75
	4	20	19	95
	Mean	20.0	16.3	81

 

Cumulative Survival and Mortality on Study Day 34 (PHD 30)of the Controland the Limit Loading Rate

Nominal test item loading rate [mg/L]	Rep.	Vital larvae on study day 34 (PHD 30)	Overall survival [%]	Overall mortality [%]
Control	1	17	85	15
	2	15	75	25
	3	16	80	20
	4	15	75	25
	Mean	15.8	79	21
10	1	19	95	5
	2	12	60	40
	3	15	75	25
	4	19	95	5
	Mean	16.3	81	19 (-)

(-)       = No statistically significant difference from control groups

Overview ofFry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Control and the Limit Loading Rate

 

Nominal test item loading rate [mg/L]	Rep.	PHD 30 (End of exposure)
		Mean total length per fish [mm]	Mean wet weight per fish [mg]
Control	1	13.6	23.5
	2	14.6	29.3
	3	14.1	26.1
	4	13.8	27.7
	Mean	14.0	26.7
	±SD	0.435	2.47
	CV [%]	3.11	9.25
10	1	14.8	29.2
	2	14.9	34.5
	3	15.0	32.4
	4	15.2	30.8
	Mean	15.0 (-)	31.7 (-)
	±SD	0.171	2.26
	CV [%]	1.14	7.13

 

 (-)      = No statistically significant difference from control groups

 IndividualLength on Study Day 34 (PHD 30)of the Control and the Limit Loading Rate

 

Fish No.	Nominal test item loading rate [mg/L]
	Control				10
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	10.0	9.5	10.0	18.5	7.0	19.5	10.0	8.5
2	7.5	15.0	15.0	18.0	17.0	17.5	18.0	15.5
3	12.0	17.5	15.5	15.0	14.5	17.5	15.5	19.5
4	10.5	15.0	8.0	15.5	16.5	11.5	15.0	16.0
5	13.0	16.0	18.0	13.5	18.0	21.5	11.5	17.0
6	13.5	14.5	14.0	6.0	17.0	16.0	20.0	17.0
7	16.5	16.0	18.5	7.5	20.0	12.0	14.5	14.0
8	14.5	12.0	14.0	7.5	11.0	12.5	17.0	17.5
9	13.0	16.0	13.0	15.0	16.5	15.0	15.0	16.0
10	16.5	16.5	13.5	11.0	13.0	18.0	17.0	14.5
11	13.0	13.0	12.5	19.0	17.5	8.0	18.0	16.0
12	15.0	14.0	16.5	15.0	15.0	10.0	11.5	15.0
13	17.0	13.5	14.0	14.0	13.0	-	16.0	10.0
14	17.0	14.0	12.0	18.0	14.5	-	10.5	18.0
15	13.0	17.0	15.0	14.0	12.5	-	15.5	11.0
16	15.5	-	16.0	-	13.5	-	-	15.0
17	14.5	-	-	-	17.5	-	-	16.0
18	-	-	-	-	12.0	-	-	18.0
19	-	-	-	-	15.0	-	-	15.0
20	-	-	-	-	-	-	-	-
Mean	13.6	14.6	14.1	13.8	14.8	14.9	15.0	15.2
±SD	2.63	2.09	2.71	4.14	3.02	4.12	2.96	2.79
CV %	19.34	14.32	19.22	30.00	20.41	27.65	19.73	18.36

                                         - = Fish died before end of the study

  Pooled Wet Weights on Study Day 34 (PHD 30)of the Control and the Limit Loading Rate

 

Nominal test item loading rate [mg/L]	Replicate	Number of fish alive on study day 34	Pooled wet weight per replicate [mg]	Mean wet weight per fish [mg]	Mean [mg]	±SD	CV %
Control	1	17	399.9	23.5	26.7	2.47	9.25
	2	15	439.7	29.3
	3	16	418.1	26.1
	4	15	415.3	27.7
10	1	19	554.0	29.2	31.7	2.26	7.13
	2	12	413.8	34.5
	3	15	485.8	32.4
	4	19	585.9	30.8

 

Specific analysis of the limit concentration of the test item in the limit loading rate and the control was carried out via LC-MS/MS. Only the monoalkylated isomers could be quantified. The measured concentrations were either extremely low or generally even below the limit of quantification (<1 μg/L). It was concluded that these results could not be used to base test concentrations upon. As a consequence, results of the present study were based on the loading rates initially prepared.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria:
Dissolved oxygen saturation was between 62 and 99 % of the air saturation value (required: > 60 %).
Water temperature did not differ by more than  1.5 °C between test vessels or between successive days at any time during the test, and was in the recommended range for the test species (i.e. 26 °C ± 1.5 °C): 24.8 – 26.9 °C.
Hatching success was 100 % in the control group (required: ≥ 70 %).
Post-hatch survival was 79 % in the control group (required: ≥ 75 %).
Analytical measurements were performed and methods validated