1-[(2-chloro-4-nitrophenyl)azo]-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 2021-09-07 to 2021-10-12

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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-[(2-chloro-4-nitrophenyl)azo]-2-naphthol

EC Number:

220-562-2

EC Name:

1-[(2-chloro-4-nitrophenyl)azo]-2-naphthol

Cas Number:

2814-77-9

Molecular formula:

C16H10ClN3O3

IUPAC Name:

1-[(2-chloro-4-nitrophenyl)diazenyl]-2-naphthol

Test material form:

solid: particulate/powder

Details on test material:

Batch no. 9A475
Purity >98

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item:
The samples were analyzed for the test item dispersed and truly 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 truly 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. Samples were taken at the start of the exposure on day 0 and at the day of change of test vessels (study day 12).
During 7 days of exposure, one sampling interval was analyzed. Freshly prepared media (after water renewal) were taken from the test vessels of the control group and the loading rates at the start of an exposure interval on study day 0 (start of the exposure), 7, 12, 21 and 28 and at the end of the corresponding interval from the aged test media on study days 1, 8, 13, 22 and 29.
The prepared loading rates used for the water renewal of the corresponding interval were sampled from the 20 L aquaria and analyzed on study days 0, 7, 12, 21 and 28.

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 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. 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 sedimentated 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 12.

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.

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 (75% of maxiumum power: 300 W) for 10 minutes. 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 volumes 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 a homogeneous distribution of the test item and to avoid sedimentation, the prepared loadings were treated with a laboratory blender for about 1 minute 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 1 minute 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.

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

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
Acidity: 0.1 mmol/L (recent measurement 2021-08-30)
Alkalinity: 0.7 mmol/L (recent measurement 2021-08-30)
Conductivity: 167 µS/cm (recent measurement 2021-08-30)
 
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. 1000 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 93 %.

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 replicate of the first test group, the 2nd 5 eggs to the second replicate of the first test group and so on, until each replicate of all test groups contained the necessary number of eggs. Distribution was started with the control group, followed by the lowest test concentration up to the highest test concentration. The eggs were placed on heightened and fixed gauze cages remaining in the crystallization dishes to avoid direct contact to the bottom and possible test item sediment.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Test conditions

Hardness:

range: 69 to 78 mg CaCO3/L
mean: 73 mg CaCO3/L

Test temperature:

range: 25.4 - 27.1 °C
mean: 26°C

pH:

range: 7.34 to 7.88
mean: 7.66 to 7.71

Dissolved oxygen:

Not less than 60% of air saturation value.

Details on test conditions:

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

Test duration:
34 days (30 days post hatch), depending on post-hatch day 0 (study day 4)

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 12: Crystallisation dishes were used. The volume of the test media in the dishes was about 700 mL.
Days 12 to 34 (test end): Glass aquaria (22/22/18 cm) were used. The volume of the test media was about 4 L.
Test vessels were covered with transparent lids.

Transfer of juveniles:
Juveniles were gently transferred to larger aquaria on study day 12.
 
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.

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 33).
Feeding started 2 days after the beginning of hatching (on study day 5 (post-hatch day 1)). 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 Concentration (target): Not less than 60% of air saturation value.

Light intensity (target): 400 ± 200 Lux

Photoperiod:
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.

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 4, 98 % of the control larvae had hatched. Therefore, study day 4 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.

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 highest 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 12).

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:

test mat.

Basis for effect:

other: Hatching success

Effect concentrations 2

Key result

Duration:

34 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Hatching success

Effect concentrations 3

Key result

Duration:

34 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:

34 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:

34 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:

34 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:

34 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:

34 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:

34 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:

34 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 93%. Eggs were fully covered with the respective test solutions during the fertilization check. 135 eggs were introduced in the control media and 13 of these eggs were discarded. 135 eggs were introduced in the loading rate 1.00 mg/L and 11 of these eggs were discarded. 133 eggs were introduced in the loading rate 3.16 mg/L and 6 of these eggs were discarded. 139 eggs were introduced in the loading rate 10.0 mg/L and 8 of these eggs were discarded. In total 38 eggs of 542 introduced eggs were discarded, resulting in a fertilization rate of 93%.

Hatching and Definition of Post Hatch Day 0:
Hatching began on study day 2 in the nominal loading rates of 1.00, and 10.0 mg/L of the test item and continued until study day 5, where the last hatched larvae in the control and the loading rates of 1.00 and 10.0 mg/L were observed. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching success of 98% in the control.

Statistical procedures were applied for the total number of test organisms that have hatched. The Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm was performed for statistical analysis of hatching success on study day 4. The Dunnett`s Multiple t-test Procedure was performed for statistical analysis of hatching success on study day 5.
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.

Swim-up:
Swim-up was observed for a 4 day period from study days 4 to 7 (see Table 4). Newly hatched fry began to swim up on study day 4 (PHD 0). On study day 7 (PHD 3), 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 86% 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 79 to 86%
The Dunnett`s Multiple t-test Procedure was performed for statistical analysis of post hatch survival data on study day 34 (PHD 30). No statistically significant differences were found for each tested loading rate up to 10 mg/L.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal loading rate), respectively.

Overall Survival:
The overall survival at the end of the exposure, related to the number of eggs introduced on day 0 was 85% in the control group and 76 to 85% in the loading rates 1.00 to 10.0 mg/L of the test item.
The Dunnett`s Multiple t-test Procedure was performed for statistical analysis of overall survival data on study day 34 (PHD 30). No statistically significant differences were found for each tested loading rate up to 10 mg/L.
The NOELR and the LOELR for this endpoint were determined to be 10 and > 10 mg/L (nominal loading rate), respectively.

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 data of fresh weight and mean total length of all survivors (Dunnett`s Multiple t-test), all tested 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 exposure.
The maximum biomass at the end of the exposure was determined in replicate 3 of the loading rate 1.00 mg/L: 641.6 mg total fish weight. The maximum biomass loading based on the 4 liter volume of a single growth chamber was 160 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 and behavioral effects were observed in the control and the nominal loading rate 1.00 mg/L. In the nominal loading rate 3.16, isolated events of arresting on the ground (G) and Quiescence (Q) were observed on study day 19. In the nominal loading rate 10.0 mg/L, isolated events of no food uptake (F), arresting on the ground (G) and a missing escape reflex were observed on study day 6.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Pigment Red 4 caused no adverse effects on Zebrafish in an early life stage toxicity test, 30 days post hatch when exposed to a nominal loading rate of 1 to 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.

Executive summary:

Pigment Red 4 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 (small 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 included 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.

 

The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each 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 four, 98 % of the control larvae had hatched. Therefore, study day 4 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 Pigment Red 4 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, 12, 21 and 28 and from corresponding 24 hours aged test media on study days 1, 8, 13, 22 and 29.

 

As the test item disperses, the effect values are considered to be based on the nominal loading rates of 1.00 – 3.16 – 10.0 mg/L, corresponding to the geometric mean measured concentrations of the dispersed fraction of the test item 0.340 – 1.16 – 2.75 mg/L and the geometric mean measured concentrations of the dissolved fraction of the test item 0.0200 – 0.0401 – 0.0413 mg/L, respectively.

 

All effect levels were given based on the geometric mean measured concentrations of the total 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 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.