1-(4-methyl-2-nitrophenylazo)-2-naphthol

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:

from 2020-06-18 to 2020-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

1-(4-methyl-2-nitrophenylazo)-2-naphthol

EC Number:

219-372-2

EC Name:

1-(4-methyl-2-nitrophenylazo)-2-naphthol

Cas Number:

2425-85-6

Molecular formula:

C17H13N3O3

IUPAC Name:

1-(4-methyl-2-nitrophenylazo)-2-naphthol

Test material form:

other: Red powder

Details on test material:

Test item Hansa Scarlet RNC-IN

Batch number INB 3100014

Purity of main component 98.8% (w/w) (comp. 1)

Chemical name 1-(4-Methyl-2nitrophenylazo)-2-naphtol (EINECS/REACH)

CAS-Name No 2425-85-6

EINECS / EC No 219-372-2

C.I. Index Pigment Red 3 (P.R.3)

Composition Pigment Red 3 98.8% (w/w)

Molecular formula C17H13N3O3

Structure






Particle size D50 = 259 nm

Molecular weight 307.31 g/mol

Density 1.4073 g/cm3

Appearance Red, powder

Water Solubility Approx. 3 µg/L

Expiry date 2021-03-03

Recommended storage Room temperature

Specific details on test material used for the study:

Test item Hansa Scarlet RNC-IN

Batch number INB 3100014

Purity of main component 98.8% (w/w) (comp. 1)

Chemical name 1-(4-Methyl-2nitrophenylazo)-2-naphtol (EINECS/REACH)

CAS-Name No 2425-85-6

EINECS / EC No 219-372-2

C.I. Index Pigment Red 3 (P.R.3)

Composition Pigment Red 3 98.8% (w/w)

Molecular formula C17H13N3O3

Structure






Particle size D50 = 259 nm

Molecular weight 307.31 g/mol

Density 1.4073 g/cm3

Appearance Red, powder
 
Water Solubility Approx. 3 µg/L

Expiry date 2021-03-03

Recommended storage Room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The samples were analyzed for the test item dispersed and genuinely dissolved in dilution water with an LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. Samples for determination of the genuinely dissolved test item (dissolved fraction) were centrifuged as specified below prior to analysis. The method was validated.

Sampling schedule
Samples of test media (including control group) were taken from alternating test replicates. The test item Hansa Scarlet RNC-IN 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.

Sampling for the analytical monitoring
For each sampling date and sample of test media at least two aliquots were taken after preparation of the test loadings and the control. Dimethyl sulfoxide was added to one replicate. One replicate was taken without addition of solvent. It was centrifuged for 15 minutes at 10000G and analyzed. The remaining replicates were stored at ambient conditions

Test solutions

Vehicle:

no

Details on test solutions:

Test design
A semi-static test procedure with daily renewal of the test media was performed. Daily renewal was chosen, due to the outcome of the experiments on dispersion stability carried out referring to OECD 318 and Series on the Safety of Manufactured Nanomaterials No. 40 Guidance Document. Eggs were exposed to the test item in test vessels filled with a defined amount of the test media (specifications see below). 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 were gently removed by siphoning with glass tubes. Care was taken not to remove or injure the test organisms. Freshly prepared media were gently added with glass tubes to the test vessels to avoid stressing of the test organisms. Any sedimented test item was daily removed during cleaning as specified below.
Fresh media for water renewals were aerated during renewal procedure for homogenization and to prevent sedimentation of the test item.
Due to the increase of growth of fish during the study, the test vessels were changed once on study day 18 depending on the life-stage and growth of fish.

Loading rates
The study was performed with three nominal loading rates of 10.0, 3.16 and 1.00 mg test item/L (dilution factor √10). According to the guideline the highest loading rate did not exceed 10 mg/L. Three loading rates were tested for profound estimation of the NOELR and LOELR values.
The loading rates are based on the results of a preliminary range finding test (see Annex II).

Preparation of the stock dispersions, application and dispersion treatment
The preparation procedure was carried out referring to UBA Texte 06/2013.
Three stock dispersions were prepared per loading rate with a total volume of 400 mL.
Stock dispersions of 500 mg/L (200 mg / 400 mL), 158 mg/L (63.2 mg / 400 mL) and 50.0 mg/L (20.0 mg / 400 mL) were prepared in dechlorinated tap water in a 500 mL flask. The dispersions were treated with sonication (300 W) for 1 hour. Thereafter, the test loading rates of 10.0, 3.16 and 1.00 mg/L were prepared with a total volume of 20 L by dilution. The total volume of 400 mL of the homogenous dispersions (stock dispersions) were filled up with dilution water to a total volume of 20 L in glass aquaria (500 mg/L for the loading rate of 10.0 mg/L, 158 mg/L for the loading rate of 3.16 mg/L and 50.0 mg/L for the loading rate of 1.00 mg/L).
To achieve homogeneous distribution of the test item and to avoid sedimentation, the prepared loadings were treated with a laboratory blender for about 3 – 5 minutes with 17000 rpm. The water column in the 20L aquaria was agitated by two flow circulation pumps, placed in two transversely opposite bottom corners of the aquaria for approx. 24 ± 2 hours. Thereafter and prior to adding the test loadings to the test vessels (where the eggs/fish were exposed) the loadings were treated again with the laboratory blender for about 3 – 5 minutes with 17000 rpm.
Fresh media for water renewals were aerated during renewal procedure for homogenization and to prevent sedimentation of the test item.
The homogeneous stock dispersions and the test loadings (in 20 L aquaria) were prepared at least 24 ± 2 hours prior to the start of the exposure and daily thereafter until the end of the exposure.

Control Dilution water (without test item)

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test organism
Danio rerio (zebrafish) Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae

Reason for the selection
According to the guideline Danio rerio is recommended for this type of study.

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


Water Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
Alkalinity: 0.6 mmol/L (recent measurement: 2020-06-17)
Acidity: 0.1 mmol/L (recent measurement: 2020-06-17)
Conductivity: 143 µS/cm (recent measurement: 2020-06-17)

Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days before the start of the exposure, 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. 800 eggs were taken and washed in dilution water. Eggs originated from 2 different spawnings. At least 70% of the eggs appeared healthy.

Start of exposure
The 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
Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and were discarded.

Fertilization rate The fertilization rate was 85 %.

Introduction of eggs
Only fertilized eggs with more than 2 cells were placed directly in the test vessels. 20 eggs were introduced by random per replicate (corresponding to 80 eggs per treatment group). Eggs were less than 24 h old at experimental starting. The distribution of eggs to the loading groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contain the necessary number of eggs.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

33 days (30 days post hatch), depending on post-hatch day 0 (study day 3)

Test conditions

Hardness:

Total hardness of the test media was measured from the control and the highest loading rate of the test item (10.0 mg/L). Total hardness was measured once per week. The mean total hardness was 64 mg CaCO3/L in the control and ranged from 59 to 72 mg CaCO3/L in the control and the highest loading rate of 10.0 mg test item/L (for details, see Table 23).

Total Hardness in the Control and the Loading Rate 10.0 mg/L

Study day Replicate Total hardness [mg CaCO3/L]
Nominal loading rate of the test item [mg/L]
Control 10.0
1 1 62 64
7 2 60 62
14 3 59 64
22 4 70 72
28 1 67 66
Mean 64 66
± SD 4.72 3.85
Min. 59 62
Max. 70 72

Min./Max. = Minimum/Maximum measured hardness

Test temperature:

Water Temperature

During the exposure the water temperature was recorded continuously (once per hour) with a data logger. The mean temperature was 26.2 °C. The minimum temperature was 25.0 °C and the maximum temperature was 27.3 °C. The water temperature did not differ by more than ± 1.5 °C between successive days during the test. The validity criterion on the parameter temperature was fulfilled.
The mean water temperature measured once per week from all replicates during the exposure period was 26.3 °C for the loading rates 1.00 and 10.0 mg/L and for the control and 26.2 °C for the loading rate 3.16 mg/L. The minimum and maximum measured temperature for all groups were 25.0 and 27.2 °C, respectively.

Water Temperature (Continuous Measuring) of the Control Group

Period of measurements 2020-06-18 to 2020-07-21
Minimum temperature [°C] 25.0
Maximum temperature [°C] 27.3
Mean temperature [°C] 26.2

pH:

pH-Values

The mean pH-values in the control and the loading rates 1.00 to 10.0 mg/L were 7.44 to 7.49 and ranged from 7.23 to 7.82 during the exposure period.

pH-Values in the Test Media (Study Day 0 – 22)

Study day Replicate Media pH
Nominal loading rate of the test item [mg/L]
Control 1.00 3.16 10.0
0 1 Fresh 7.30 7.41 7.40 7.35
2 7.28 7.39 7.37 7.33
3 7.28 7.38 7.33 7.34
4 7.37 7.38 7.32 7.31
1 1 Aged 7.57 7.60 7.70 7.72
2 7.61 7.58 7.66 7.58
3 7.61 7.59 7.65 7.58
4 7.58 7.55 7.68 7.55
7 1 Fresh 7.23 7.28 7.32 7.31
2 7.26 7.26 7.27 7.27
3 7.23 7.24 7.27 7.25
4 7.25 7.26 7.24 7.24
8 1 Aged 7.64 7.51 7.62 7.52
2 7.54 7.51 7.50 7.47
3 7.55 7.56 7.48 7.48
4 7.53 7.59 7.52 7.47
14 1 Fresh 7.35 7.32 7.28 7.34
2 7.34 7.29 7.26 7.31
3 7.29 7.25 7.29 7.26
4 7.27 7.28 7.30 7.28
15 1 Aged 7.24 7.44 7.47 7.45
2 7.29 7.43 7.46 7.42
3 7.36 7.44 7.44 7.43
4 7.39 7.44 7.44 7.40
21 1 Fresh 7.38 7.46 7.48 7.52
2 7.34 7.45 7.46 7.48
3 7.39 7.45 7.47 7.48
4 7.41 7.46 7.49 7.49
22 1 Aged 7.75 7.79 7.80 7.82
2 7.67 7.81 7.78 7.80
3 7.73 7.78 7.79 7.81
4 7.74 7.78 7.82 7.82

Min./Max. = Minimum/Maximum measured pH-value
 
pH-Values in the Test Media (Study Day 28 – 29)

Study day Replicate Media pH
Nominal loading rate of the test item [mg/L]
Control 1.00 3.16 10.0
28 1 Fresh 7.34 7.36 7.36 7.33
2 7.33 7.36 7.36 7.32
3 7.34 7.36 7.38 7.33
4 7.34 7.35 7.39 7.35
29 1 Aged 7.56 7.76 7.73 7.62
2 7.68 7.70 7.71 7.64
3 7.71 7.60 7.70 7.66
4 7.69 7.73 7.65 7.69
Mean 7.44 7.48 7.49 7.47
± SD 0.168 0.166 0.174 0.170
Min. 7.23 7.24 7.24 7.24
Max. 7.75 7.81 7.82 7.82

Min./Max. = Minimum/Maximum measured pH value

Dissolved oxygen:

Dissolved Oxygen

The dissolved oxygen concentrations in the control and the test item groups, expressed in percent saturation, were in the mean 90 to 91% for all test groups and the control and ranged from 61 to 100% during the exposure period.

For more details see window below "Any other information on rsults incl. tables ".

Nominal and measured concentrations:

The study was performed with three nominal loading rates of 10.0, 3.16 and 1.00 mg test item/L (dilution factor √10).
The samples of the test item Hansa Scarlet RNC-IN 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), 7, 14, 21 and 28 and from corresponding 24 hours aged test media on study days 1, 8, 15, 22 and 29 (end of the exposure). Freshly prepared test media, which were used for the replacement of about 75% old media, was analyzed weekly. Details of the analytical method are presented in part 11. The test item concentrations of Hansa Scarlet RNC-IN and the control were analytically verified via LC-MS/MS. The freshly prepared stock solutions were analyzed once per week. The medium of the start of the exposure and the corresponding end of the exposure was analytical verified once per week. For all samples the dispersed fraction was analyzed by dilution of the sample with DMSO directly after sampling. Additional the dissolved fraction was analyzed after centrifugation of the samples to remove undissolved test item. For all determinations the geometric mean measured concentrations were calculated.

For further information see field below "Any other information on materials and methods incl. tables "

Details on test conditions:

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

Test duration 33 days (30 days post hatch), depending on post-hatch day 0 (study day 3)

Replicates, number of eggs
Four replicates per loading rate and control, with 20 eggs each (80 eggs per loading rate and control)
For the whole study 320 eggs/fish were used.

Test vessels Days 0 to 18:
Crystallisation dishes were used. The volume of the test media in the dishes was about 700 mL.

Days 18 to test end:
Glass aquaria (12.5 x 11 x 18 cm) were used. The volume of the test media was about 2.5 L.

Test vessels were covered with transparent lids.

Transfer of juveniles Juveniles were gently transferred to larger aquaria on study day 18.
 
Loading
A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.

Cleaning
The test vessels were siphoned at least once daily (during water renewal) to remove possible sediment of the test item, excess fecal matter and uneaten food, also to minimize microbial growth and biodegradation of the test item. Cleaning started on study day 1 (post hatch day -2).

Aeration
The dilution water used for preparation of the test media was aerated. A gentle aeration was applied for all test vessels.

Dilution water
Same as used for holding.

Feeding of test fish The feeding regime was ad libitum during the whole feeding period (study day 5 to 32).
Feeding started 3 days after the beginning of hatching (on study day 5 (post-hatch day 2)). 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).
Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless steel mesh and resuspended in tap water. Feeding ad libitum was carried out.

Water temperature (target) 26 ± 1.5 °C

Dissolved oxygen Not less than 60% of air saturation value.
Concentration (target)

Light intensity (target) 400 ± 200 Lux

Photoperiod A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
 
Type and Frequency of Measurements
Photographic Documentation

During exposure the character of the stock dispersions, test loadings (freshly prepared and aged) and appearance of eggs and embryos (i.e. deposition of the test item) were documented via photographs where appropriate. Attention is paid to the homogeneity of the aqueous media (e.g. sedimentation) and the adhesion of the test item onto the embryonic membrane. Pictures were taken daily during the first 5 days of exposure (time to hatch) and weekly thereafter until the end of the study.

Biological Parameters

All biological parameters were observed daily. Dead larvae/fish and coagulated or dead eggs were removed daily, if observed as described below.

Hatching
The number of hatched larvae was determined daily until study day 5. Eggs were only removed, when mortality of eggs/embryos was observed as specified below.
On study day 3, 96 % of the control larvae had hatched. Therefore, study day 3 was defined as post-hatch day 0 (= PHD 0). For evaluation of hatch, all hatched larvae (even dead ones) were counted.
The cumulative number of hatched larvae was used for evaluation. For further details on evaluation see part 5.

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.

Physical Properties

Water quality measurements were carried out during exposure in the following intervals:

Once per hour Temperature in the dilution water, measured in a separate vessel filled with dilution water

Daily Determination of
- Dissolved oxygen in all replicates of each test group from fresh and old test media

Once per week
Determination of
-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
- TOC and Chlorine from the dilution water
- Total hardness in one replicate of the control group and the limit loading rate

The light intensity on the surface of the test aquaria was measured at the start of the exposure and on the day of changing the test vessels (study day 18).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Effect concentrations 2

Key result

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Effect concentrations 3

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 4

Key result

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 5

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 6

Key result

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 7

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Effect concentrations 8

Key result

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Effect concentrations 9

Key result

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall survival

Effect concentrations 10

Key result

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall survival

Details on results:

Egg Fertilization Rate

The egg fertilization rate, determined on study day 0 (start of the exposure) was 85 %.
Eggs were fully covered with the respective test solutions during fertilization check.
126 eggs were introduced in the control media and 21 of these eggs were discarded.
125 eggs were introduced in the loading rate 1.00 mg/L and 20 of these eggs were discarded.
120 eggs were introduced in the loading rate 3.16 mg/L and 14 of these eggs were discarded.
126 eggs were introduced in the loading rate 10.0 mg/L and 19 of these eggs were discarded.
In total 74 eggs of 497 introduced eggs were discarded, resulting in a fertilization rate of 85%.

Hatching and Definition of Post Hatch Day 0

Hatching began on study day 2 in the control and the loading rates 1.00, 3.16 and 10.0 mg/L of the test item and continued until study day 4, where the last hatched larvae in the loading of 1.00 mg/L was observed. Study day 3 was determined to be post hatch day 0 (PHD 0) with a hatching success of 96% in the control and in the loading rates 1.00 and 3.16 mg/L and 90% in the loading rate 10.0 mg/L. Hatching was completed in the loading rate 1.00 mg/L at study day 4 (100% hatching success).

Statistical procedures were applied for the total number of test organisms that have hatched. The Chi2 2x2 Table Test with Bonferroni Correction was performed for statistical analysis of hatching success on study day 3 and 6. No statistically significant difference was found between the control and each loading rate.
The NOELR and the LOELR (nominal loading rate) for this endpoint were determined to be 10 mg/L and > 10 mg/L, respectively.
Hatching Success in the Control and the Loading Rates
Nominal
loading rate
of the test item
[mg/L] Rep. PHD -2 PHD -1 PHD 0 PHD 1
Study day 1 Study day 2 Study day 3 Study day 4
Hatching success [%] per study day
Control 1 0 0 100 100
2 0 0 90 90
3 0 0 100 100
4 0 0 95 95
Mean 0 0 96 96
1.00 1 0 0 100 100
2 0 0 95 100
3 0 0 100 100
4 0 0 90 100
Mean 0 0 96 (-) 100
3.16 1 0 0 85 85
2 0 0 100 100
3 0 0 100 100
4 0 0 100 100
Mean 0 0 96 (-) 96
10.0 1 0 0 100 100
2 0 0 75 75
3 0 0 95 95
4 0 0 90 90
Mean 0 0 90 (-) 90

(-) = No statistically significant difference from control group

Swim-up

Swim-up was observed for a 4 day period from study days 4 to 7. Newly hatched fry began to swim up on study day 4 (PHD 1). On study day 7 (PHD 4), all surviving larvae had swum up, except one larvae in the control, which swam in lateral position, showed coordination disorders and died on study day 11. No statistical analysis of swim-up data was carried out.

Percent Swim-up of Hatched Live Fry of the Control and the Loading Rates
Nominal
loading rate
of the test item
[mg/L] Rep. PHD 0 PHD 1 PHD 2 PHD 3 PHD 4
Study day 3 Study day 4 Study day 5 Study day 6 Study day 7
Swim up [%]
Control 1 0 10 100 100 100
2 0 6 83 94 94
3 0 5 100 100 100
4 0 5 100 100 100
Mean 0 6 96 99 99
1.00 1 0 5 100 100 100
2 0 0 100 100 100
3 0 0 90 100 100
4 0 0 90 100 100
Mean 0 1 95 100 100
3.16 1 0 0 75 100 100
2 0 0 95 100 100
3 0 0 85 95 100
4 0 0 100 100 100
Mean 0 0 89 99 100
10.0 1 0 0 100 100 100
2 0 0 100 100 100
3 0 0 95 100 100
4 0 6 94 100 100
Mean 0 1 97 100 100

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 78% in the control. The post-hatch survival in the loading rates 1.00 to 10.0 mg/L of the test item was in the range of 76 to 82%.

The Chi2 2x2 Table Test with Bonferroni Correction was performed for statistical analysis of post hatch survival data on study day 33 (PHD 30). No statistically significant differences were found for each loading rate of 10 mg/L.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal loading rate), respectively.
The LLR0 value for post hatch survival on study day 33 (PHD 30) was determined to be 10 mg/L (nominal limit loading rate).

Post-Hatch Survival on Study Day 33 (PHD 30) of the Control and the Loading Rates
Nominal
loading rate
of the test item
[mg/L] Rep. Eggs introduced on study day 0 Cumulative number of hatched larvae Vital Larvae on
study day 33
(PHD 30)
Post-Hatch survival
on study day 33

[%]
Control 1 20 20 16 80
2 20 18 12 67
3 20 20 18 90
4 20 19 14 74
Mean 20 19 15 78
1.00 1 20 20 15 75
2 20 20 15 75
3 20 20 18 90
4 20 20 13 65
Mean 20 20 15 76 (-)
3.16 1 20 17 13 76
2 20 20 15 75
3 20 20 19 95
4 20 20 16 80
Mean 20 19 16 82 (-)
10.0 1 20 20 15 75
2 20 15 13 87
3 20 19 16 84
4 20 18 13 72
Mean 20 18 14 80 (-)

(-) = No statistically significant difference from control groups

Overall Survival

The overall survival at the end of the exposure, related to the number of eggs introduced on day 0 was 75% in the control group and 71 to 79% in the loading rates 1.00 to 10.0 mg/L of the test item.

The Chi2 2x2 Table Test with Bonferroni Correction was performed for statistical analysis of overall survival data on study day 33 (PHD 30). No statistically significant differences were found for each nominal loading rate.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal loading rate), respectively.
The LLR0 value for overall survival on study day 33 (PHD 30) was determined to be 10 mg/L (nominal loading rate).

Cumulative Survival and Mortality on Study Day 33 (PHD 30) of the Control and the Limit Loading Rate

Nominal
loading rate
of the test item
[mg/L] Rep.
Vital larvae on study day 33
(PHD 30)
Overall survival [%] Overall mortality [%]
Control 1 16 80 20
2 12 60 40
3 18 90 10
4 14 70 30
Mean 15 75 25
1.00 1 15 75 25
2 15 75 25
3 18 90 10
4 13 65 35
Mean 15 76 24 (-)
3.16 1 13 65 35
2 15 75 25
3 19 95 5
4 16 80 20
Mean 16 79 21 (-)
10.0 1 15 75 25
2 13 65 35
3 16 80 20
4 13 65 35
Mean 14 71 29 (-)

(-) = No statistically significant difference from control groups
Fry Growth

Fry growth, expressed as length and wet weight, was measured on study day 33 (PHD 30) from all survivors.
With the statistical procedures applied for fresh weight data and mean total length of all survivors (Dunnett`s Multiple t-test) all nominal loading rates showed no significant differences for these parameters. Therefore the NOELR and the LOELR for both parameters are 10 mg/L and > 10 mg/L (nominal loading rate).

Biomass Loading

The biomass-loading factor for the study was determined from the fresh weight of the control fish and the fish of the loading rates at the end of the exposur.

The maximum biomass at the end of the exposure was determined in replicate 3 of the loading rate 1.00 mg/L: 557.3 mg total fish weight. The maximum biomass loading based on the 2.5 liter volume of a single growth chamber was 223 mg/L.

Maximum loading rate: biomass/(volume of test solution) = (557.3 mg)/(2.5 L) = 223 mg/L

This loading was well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
 
Overview of Fry Growth: Length and Wet Weight on Study Day 33 (PHD 30) of the Control and the Loading Rates

Nominal
loading rate
of the test item
[mg/L] Rep. PHD 30 (End of exposure)
Mean total length per fish
[mm] Mean wet weight per fish
[mg]
Control 1 14.8 26.7
2 15.6 33.4
3 14.4 26.3
4 15.2 31.2
Mean 15.0 29.4
± SD 0.462 3.01
CV [%] 3.09 10.2
1.00 1 15.1 30.4
2 15.1 29.2
3 15.0 31.0
4 14.9 32.4
Mean 15.0 (-) 30.7 (-)
± SD 0.0677 1.18
CV [%] 0.451 3.83
3.16 1 15.0 29.6
2 14.6 29.0
3 13.7 25.0
4 14.5 29.5
Mean 14.4 (-) 28.3 (-)
± SD 0.445 1.91
CV [%] 3.08 6.75
10.0 1 14.9 31.1
2 15.8 36.7
3 14.0 28.5
4 15.7 37.1
Mean 15.1 (-) 33.4 (-)
± SD 0.706 3.68
CV [%] 4.68 11.0

(-) = No statistically significant difference from control groups
Individual Length on Study Day 33 (PHD 30) of the Control and the Loading Rates

Fish No. Nominal loading rate of the test item
[mg/L]
Control 1.00 3.16 10.0
Total length of individual fish in [mm]
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 16.5 15.5 14.0 17.5 15.0 15.0 13.0 18.0 15.0 18.5 14.0 14.5 15.0 17.0 11.0 15.0
2 16.0 16.0 13.0 12.0 14.5 16.0 13.0 13.0 16.0 13.0 14.0 14.0 14.0 13.5 15.0 18.0
3 15.0 17.0 15.0 14.5 14.5 16.5 13.0 15.0 15.0 14.5 15.0 13.0 13.0 15.5 14.0 17.5
4 15.0 16.5 17.5 15.0 15.5 17.0 14.5 10.5 16.0 15.0 14.0 14.0 11.0 15.5 12.0 17.0
5 16.5 16.5 17.5 17.0 14.0 12.5 14.5 12.0 16.0 15.0 14.5 11.5 15.5 16.5 12.0 15.0
6 15.0 18.0 17.0 14.5 15.0 15.5 15.0 13.0 15.0 12.0 13.0 15.0 16.5 15.5 14.5 15.0
7 14.0 15.5 13.5 12.5 17.0 15.5 18.0 15.0 13.0 13.0 13.5 15.5 15.0 14.0 15.0 15.0
8 16.5 10.5 10.0 14.0 14.0 13.0 16.0 19.0 10.0 12.0 14.0 16.0 14.0 17.5 13.0 15.5
9 18.0 17.0 12.0 13.0 15.5 14.5 16.5 16.0 12.0 15.5 14.0 12.0 15.5 16.0 10.5 16.0
10 11.0 19.5 16.5 18.0 15.0 16.5 16.0 17.0 14.0 14.5 11.0 14.5 15.5 15.5 16.5 14.0
11 15.5 12.5 16.0 16.0 12.5 17.0 16.0 13.0 17.5 15.0 16.0 15.0 15.0 14.5 17.0 16.0
12 14.0 12.5 15.0 15.5 18.0 14.0 14.5 17.0 18.0 16.0 15.0 14.0 14.5 15.0 16.0 15.0
13 11.0 ̶ 8.0 17.0 19.5 14.0 16.0 15.5 17.0 15.5 10.0 16.5 15.5 19.0 16.0 15.0
14 10.0 ̶ 15.5 16.5 16.5 13.0 15.5 ̶ ̶ 14.0 13.5 15.5 15.0 ̶ 12.0 ̶
15 16.5 ̶ 15.0 ̶ 10.0 16.0 17.0 ̶ ̶ 15.0 15.0 15.5 18.0 ̶ 15.0 ̶
16 15.5 ̶ 15.0 ̶ ̶ ̶ 16.0 ̶ ̶ ̶ 14.0 16.0 ̶ ̶ 15.0 ̶
17 ̶ ̶ 14.5 ̶ ̶ ̶ 14.0 ̶ ̶ ̶ 15.0 ̶ ̶ ̶̶ ̶ ̶
18 ̶ ̶ 13.5 ̶ ̶ ̶ 11.5 ̶ ̶ ̶ 13.5 ̶ ̶ ̶ ̶ ̶
19 ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ 12.0 ̶ ̶ ̶ ̶ ̶
20 ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶
Mean 14.8 15.6 14.4 15.2 15.1 15.1 15.0 14.9 15.0 14.6 13.7 14.5 14.9 15.8 14.0 15.7
± SD 2.20 2.45 2.42 1.83 2.15 1.45 1.61 2.41 2.17 1.60 1.41 1.37 1.51 1.42 1.96 1.12
CV % 14.92 15.71 16.85 12.02 14.27 9.61 10.72 16.14 14.51 10.99 10.25 9.46 10.2 9.0 14.0 7.1
- = Fish died before end of the study
 
Pooled Wet Weights on Study Day 33 (PHD 30) of the Control and the Loading Rates

Nominal
loading rate
of the test item
[mg/L] Replicate

Number of fish alive
on study day 33 Pooled
wet weight
per replicate
[mg] Mean
wet weight
per fish
[mg] Mean


[mg] ± SD


CV %



Control 1 16 426.8 26.7 29.4 3.01 10.2
2 12 400.3 33.4
3 18 473.0 26.3
4 14 437.3 31.2
1.00 1 15 455.5 30.4 30.7 1.18 3.83
2 15 437.3 29.2
3 18 557.3 31.0
4 13 421.5 32.4
3.16 1 13 384.5 29.6 28.3 1.91 6.75
2 15 435.0 29.0
3 19 474.4 25.0
4 16 471.4 29.5
10.0 1 15 466.7 31.1 33.4 3.68 11.0
2 13 477.0 36.7
3 16 455.7 28.5
4 13 482.6 37.1


Morphological and Behavioral Effects

No biologically significant morphological and behavioral effects were observed in the nominal loading rate 3.16 mg/L. In the control and the nominal loading rates 1.00 and 10.0 mg/L, isolated events of side position (S), coordination issues (C) and Quiescence (Q) were observed from study day 7 up to study day 21.

Behavioral Effects of the Control and the Nominal Loading Rates 1.00 and 10.0 mg/L (Study Day 7 – 21)

Study Day Control 1.00 mg/L 10.0 mg/L
1 2 3 4 1 2 3 4 1 2 3 4
7 - 1: S - - - - - - - - - -
8 - 1: S - - - - - - - - - -
9 - 1: C - - - - - - - - - -
10 - 1: C - - - - - - - - - -
11 - - - - - - 1: Q - - - - -
12 - - - - - - 1: Q - - - - -
13 - - - - - - 1: Q - - - - -
14 - - - - - - - - - - - -
15 - - - - - - - - - - - -
16 - - - - - - - - - - - -
17 - - - - - - - - - - - -
18 - - - - - - - - 1: Q - 1: Q -
19 - - - - 2: Q - - - 1: Q - 1: Q -
20 - - - - - - - - 1: Q - 1: Q -
21 - - - 1: Q - - - - - - - -

S = Larvae was lying on its side
C = Coordination issues
Q = Quiescence

Any other information on results incl. tables

Dissolved Oxygen

The dissolved oxygen concentrations in the control and the test item groups, expressed in percent saturation, were in the mean 90 to 91% for all test groups and the control and ranged from 61 to 100% during the exposure period.

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 - 3)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

0       1       Fresh       91       89       87       87

      2       Fresh       89       88       87       88

      3       Fresh       87       87       87       88

      4       Fresh       87       87       87       88

1       1       Fresh       93       91       91       91

             Aged       89       86       87       86

      2       Fresh       93       91       91       89

             Aged       89       86       85       81

      3       Fresh       92       89       90       91

             Aged       89       81       85       82

      4       Fresh       91       90       90       90

             Aged       86       85       86       81

2       1       Fresh       97       94       94       93

             Aged       99       95       91       92

      2       Fresh       96       93       93       92

             Aged       97       94       92       88

      3       Fresh       95       92       93       92

             Aged       96       90       90       91

      4       Fresh       95       91       93       91

             Aged       97       92       91       89

3       1       Fresh       94       92       91       92

             Aged       99       93       90       91

      2       Fresh       93       92       92       92

             Aged       98       92       91       87

      3       Fresh       93       91       92       91

             Aged       97       82       90       87

      4       Fresh       93       91       92       92

             Aged       96       87       91       89

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 4 - 8)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

4       1       Fresh       95       94       92       92

             Aged       100       96       93       92

      2       Fresh       94       94       94       93

             Aged       99       95       93       91

      3       Fresh       92       93       92       93

             Aged       97       92       92       91

      4       Fresh       92       92       92       92

             Aged       97       91       92       91

5       1       Fresh       96       92       92       92

             Aged       97       94       93       92

      2       Fresh       94       92       92       92

             Aged       96       92       93       93

      3       Fresh       93       91       92       92

             Aged       96       90       90       90

      4       Fresh       93       92       93       92

             Aged       96       92       92       91

6       1       Fresh       95       93       93       91

             Aged       95       93       88       90

      2       Fresh       93       93       91       91

             Aged       91       91       88       89

      3       Fresh       92       91       91       89

             Aged       91       89       86       83

      4       Fresh       92       92       90       89

             Aged       92       90       87       85

7       1       Fresh       83       87       90       88

             Aged       84       65       80       78

      2       Fresh       87       86       88       88

             Aged       75       70       78       75

      3       Fresh       89       88       88       84

             Aged       85       70       71       68

      4       Fresh       86       89       89       83

             Aged       72       75       76       64

8       1       Fresh       91       90       92       91

             Aged       92       85       89       83

      2       Fresh       91       90       91       92

             Aged       87       88       80       82

      3       Fresh       92       91       90       92

             Aged       90       90       79       81

      4       Fresh       91       91       91       91

             Aged       85       91       87       81

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 9 - 13)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

9       1       Fresh       92       93       94       92

             Aged       88       84       87       80

      2       Fresh       94       93       92       93

             Aged       89       87       86       84

      3       Fresh       94       91       92       91

             Aged       91       87       85       83

      4       Fresh       92       91       93       92

             Aged       85       91       83       80

10       1       Fresh       90       94       92       94

             Aged       91       89       90       89

      2       Fresh       98       93       93       94

             Aged       91       88       86       88

      3       Fresh       92       90       92       94

             Aged       88       86       84       86

      4       Fresh       92       91       95       96

             Aged       88       88       85       87

11       1       Fresh       93       95       93       95

             Aged       97       93       92       92

      2       Fresh       94       94       93       95

             Aged       96       91       92       93

      3       Fresh       95       91       90       95

             Aged       96       87       84       90

      4       Fresh       95       94       93       96

             Aged       94       92       89       91

12       1       Fresh       86       89       92       92

             Aged       91       88       91       88

      2       Fresh       90       89       90       89

             Aged       87       88       90       84

      3       Fresh       89       87       89       90

             Aged       91       86       88       85

      4       Fresh       89       90       89       91

             Aged       88       88       90       88

13       1       Fresh       83       91       92       90

             Aged       86       90       91       86

      2       Fresh       87       90       92       90

             Aged       90       90       86       75

      3       Fresh       91       83       87       90

             Aged       94       85       84       84

      4       Fresh       89       88       91       90

             Aged       92       90       87       83

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 14 - 18)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

14       1       Fresh       87       88       87       90

             Aged       91       85       87       89

      2       Fresh       85       88       87       90

             Aged       90       89       87       85

      3       Fresh       90       87       90       89

             Aged       94       91       81       86

      4       Fresh       86       87       88       88

             Aged       92       90       84       84

15       1       Fresh       87       88       84       88

             Aged       83       86       87       86

      2       Fresh       86       90       85       89

             Aged       87       88       85       85

      3       Fresh       90       90       86       89

             Aged       93       90       84       85

      4       Fresh       87       87       86       87

             Aged       90       88       87       81

16       1       Fresh       90       89       91       90

             Aged       92       86       91       88

      2       Fresh       89       90       90       86

             Aged       87       89       87       86

      3       Fresh       90       89       92       86

             Aged       92       91       89       85

      4       Fresh       86       93       90       87

             Aged       87       91       87       86

17       1       Fresh       72       83       90       90

             Aged       75       71       67       70

      2       Fresh       75       84       87       89

             Aged       73       69       74       69

      3       Fresh       80       87       89       86

             Aged       69       78       75       68

      4       Fresh       71       85       84       86

             Aged       64       77       65       63

18       1       Fresh       95       92       92       91

             Aged       85       85       86       83

      2       Fresh       94       91       92       91

             Aged       83       86       86       91

      3       Fresh       94       92       90       89

             Aged       88       84       86       75

      4       Fresh       93       92       90       91

             Aged       82       85       90       76

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 19 - 23)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

19       1       Fresh       97       96       92       94

             Aged       94       86       82       89

      2       Fresh       95       95       91       92

             Aged       92       86       84       63

      3       Fresh       95       94       93       92

             Aged       93       84       79       61

      4       Fresh       94       95       92       92

             Aged       85       87       88       71

20       1       Fresh       90       92       90       91

             Aged       91       94       89       93

      2       Fresh       90       92       92       92

             Aged       94       93       87       91

      3       Fresh       91       91       92       91

             Aged       94       91       91       91

      4       Fresh       90       91       90       90

             Aged       94       94       89       91

21       1       Fresh       84       93       90       93

             Aged       89       92       83       91

      2       Fresh       87       92       93       93

             Aged       88       92       89       92

      3       Fresh       90       89       93       95

             Aged       91       86       91       93

      4       Fresh       89       91       91       93

             Aged       91       89       91       91

22       1       Fresh       90       90       90       92

             Aged       86       90       84       88

      2       Fresh       92       92       92       93

             Aged       86       92       90       90

      3       Fresh       89       94       91       93

             Aged       89       88       89       92

      4       Fresh       91       91       91       92

             Aged       88       88       92       92

23       1       Fresh       91       100       100       99

             Aged       97       100       100       97

      2       Fresh       95       100       100       100

             Aged       98       100       100       98

      3       Fresh       98       100       99       99

             Aged       100       100       99       100

      4       Fresh       97       99       100       100

             Aged       98       99       100       100

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 24 - 28)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

24       1       Fresh       95       91       92       96

             Aged       94       90       84       97

      2       Fresh       93       91       93       97

             Aged       89       87       87       94

      3       Fresh       94       90       94       95

             Aged       87       89       90       92

      4       Fresh       92       93       94       94

             Aged       90       91       90       90

25       1       Fresh       93       92       95       93

             Aged       95       94       95       93

      2       Fresh       93       93       96       93

             Aged       96       96       94       92

      3       Fresh       93       92       94       94

             Aged       97       96       94       95

      4       Fresh       87       91       95       98

             Aged       94       91       96       95

26       1       Fresh       90       89       92       91

             Aged       88       90       94       87

      2       Fresh       89       88       92       86

             Aged       90       90       92       81

      3       Fresh       87       86       93       92

             Aged       87       90       95       93

      4       Fresh       85       88       94       93

             Aged       83       88       96       86

27       1       Fresh       93       93       96       97

             Aged       94       89       94       95

      2       Fresh       93       96       96       97

             Aged       96       94       94       95

      3       Fresh       93       95       97       97

             Aged       95       95       95       96

      4       Fresh       93       94       96       95

             Aged       94       92       94       95

28       1       Fresh       91       95       95       92

             Aged       88       86       90       81

      2       Fresh       94       93       95       91

             Aged       88       89       93       82

      3       Fresh       89       90       93       95

             Aged       83       90       91       80

      4       Fresh       92       95       91       96

             Aged       89       92       82       81

 

Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 29 - 33)

Study day       Replicate       Media       Dissolved oxygen [%]

                    Nominal loading rate of the test item [mg/L]

                    Control       1.00       3.16       10.0

29       1       Fresh       90       96       96       96

             Aged       89       96       95       91

      2       Fresh       95       97       96       96

             Aged       95       93       94       91

      3       Fresh       92       93       96       97

             Aged       96       88       93       93

      4       Fresh       91       95       95       97

             Aged       94       95       92       95

30       1       Fresh       95       94       96       95

             Aged       94       97       94       92

      2       Fresh       96       98       95       96

             Aged       94       96       96       92

      3       Fresh       95       95       95       97

             Aged       94       90       97       95

      4       Fresh       93       94       91       97

             Aged       91       96       97       93

31       1       Fresh       94       95       95       98

             Aged       98       98       97       96

      2       Fresh       93       93       95       96

             Aged       98       98       98       95

      3       Fresh       93       95       96       97

             Aged       97       98       99       98

      4       Fresh       92       94       96       97

             Aged       96       98       95       97

32       1       Fresh       97       95       94       97

             Aged       100       99       98       99

      2       Fresh       95       95       95       94

             Aged       98       99       98       97

      3       Fresh       90       94       95       94

             Aged       97       99       98       97

      4       Fresh       92       94       95       93

             Aged       96       98       97       96

33       1       Aged       90       92       89       91

      2       Aged       91       91       90       92

      3       Aged       92       90       90       92

      4       Aged       93       92       89       90

Mean       91       91       91       90

SD       5.31       4.98       5.08       6.53

Min.       64       65       65       61

Max.       100       100       100       100

      Min./Max.       = Minimum/Maximum measured dissolved oxygen concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Hansa Scarlet RNC-IN caused no adverse effects on Zebrafish in an early life stage toxicity test, 30 days post hatch in comparison to the dilution water control group exposed to a nominal loading rate of 10 mg test item/L.

For the parameter hatching success the NOELR was 10 mg/L. Therefore the LOELR for hatchability was determined to be > 10 mg/L. For the parameters post hatch and overall survival (mortality) the NOELR was 10 mg/L. Therefore, the LOELR for these parameters was determined to be > 10 mg/L. For the parameter fry growth (expressed as length and fresh weight) the NOELR was 10 mg/L. Therefore, the LOELR for these parameters was determined to be > 10 mg/L.
As the test item disperses, the effect values are considered to be based on the nominal loading rates.

Additionally effect levels were given based on the geometric mean measured concentrations of the dispersed fraction of the test item and on the geometric mean measured concentrations of the dissolved fraction of the test item.

Executive summary:

The effects of the test item Hansa Scarlet RNC-IN (Batch-No. INB 3100014) to the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2020-06-18 to 2020-07-21.

Hansa Scarlet RNC-IN is a red powder with a water solubility of approximately 3 µg/L. Three loading rates were tested for profound estimation of the NOELR and LOELR values. According to the guideline the highest loading rate did not exceed 10 mg/L.

With regard to the specific properties of the test item (particles, poorly water soluble, pigment), the study was carried out with dispersions of the test item (i.e. in excess of water solubility). The preparation of the dispersions include ultra-sonication and vigorous mixing to disperse the test item in water, followed by low energy mixing with circulating pumps to prevent sedimentation.

The study was conducted as a semi-static test and daily renewal of the test media. Daily renewal was chosen, due to the outcome of the preliminary experiments on dispersion stability carried out referring to OECD 318 and Series on the Safety of Manufactured Nanomaterials No. 40 Guidance Document.

The test was started by placing fertilized eggs into the test vessels and it lasted 33 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each loading rate and the control (4 replicates with 20 eggs each).

The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.

On study day three, 96 % of the control larvae had hatched. Therefore, study day 3 was defined as post hatch day 0 (= PHD 0).

Different toxicological endpoints were determined: hatching success, time to hatch, fry growth (expressed as length and fresh weight), morphological and behavioral effects, post-hatch survival and overall survival (mortality).

Specific analysis of each loading rate of Hansa Scarlet RNC-IN and the control was carried out via LC-MS/MS. The dispersed fraction of the test item was determined after addition of DMSO directly after sampling. The dissolved fraction was determined after centrifugation of the water samples, to remove undissolved test item. The concentrations were analytically verified twice during 7 days of exposure from freshly prepared test media on study day 0 (start of the exposure), 7, 14, 21 and 28 and from corresponding 24 hours aged test media on study days 1, 8, 15, 22 and 29.

 

As the test item disperses, the effect values are considered to be based on the nominal loading rates. Additionally, the specific analysis determined the ratio of truly dissolved and particulate test item.

Therefore all effect values are given based on the nominal limit loading rate of the test item. Additionally effect levels were given based on the geometric mean measured concentrations of the dispersed fraction of the test item and on the geometric mean measured concentrations of the dissolved fraction of the test item. In the samples, the sedimentated fraction was not included. However, this fraction was also present in the test vessels.

Findings and Observations

The results of the parameters hatching success, time to hatch, fry growth (expressed as weight and length), post-hatch survival and overall survival were checked for statistically significant differences.

No statistically significant differences were detected between the dilution water control and the each loading rate 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.

The effect values NOELR and LOELR were determined based on the statistical results. The results are presented in the tables below:

NOELR and LOELR Values of Hatching Success and Fry Growth

Parameter        Hatching Success        Fry Growth

expressed as:

              Length        Weight

Nominal test item loading [mg/L]

NOELR

[mg/L]        10        10        10

LOELR

[mg/L]       > 10        > 10        > 10

geometric mean measured concentrations

of the dispersed fraction of the test item [mg/L]

NOELR

[mg/L]        3.27        3.27        3.27

LOELR

[mg/L]        > 3.27        > 3.27        > 3.27

geometric mean measured concentrations

of the dissolved fraction of the test item [mg/L]

NOELR

[mg/L]        0.00581        0.00581        0.00581

LOELR

[mg/L]        > 0.00581        > 0.00581        > 0.00581

 

NOELR and LOELR Values of Post Hatch Survival and Overall Survival

Parameter        Post hatch survival        Overall survival

             

Nominal test item loading [mg/L]

NOELR

[mg/L]        10        10

LOELR

[mg/L]        > 10        > 10

geometric mean measured concentrations

of the dispersed fraction of the test item [mg/L]

NOELR

[mg/L]        3.27        3.27

LOELR

[mg/L]        > 3.27        > 3.27

geometric mean measured concentrations

of the dissolved fraction of the test item [mg/L]

NOELR

[mg/L]        0.00581        0.00581

LOELR

[mg/L]        > 0.00581        > 0.00581