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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Remarks:
with minor component
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: ASTM. 2009. Standard guide for conducting early life‐stage toxicity tests with fishes. ASTM E 1241– 05. American Society for Testing and Materials, West Conshohocken, PA, USA.
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken twice per week in all test chambers throughout the test
- Sampling method: Samples were taken by glass pipette from the center of the test tanks at mid-depth and placed in autoinjector vials or solvent-rinsed screw-cap tubes for the manually injected tests.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Analytical stock solutions were prepared by dissolving test substance in acetonitrile or methanol. All stock solutions used for testing were generated with a liquid/liquid equilibrator without using organic solvents.The solutions were subsequently transferred to the dilution system via a metering pump (fluid metering).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: obtained from Mount Lassen trout farms, Red Bluff, CA, USA and Seven Pines trout farms, Lewis, WI, USA

POST-HATCH FEEDING
- Start date: as swim-up occured (~ 45d post fertilisation)
- Type/source of feed: trout chow
- Amount given: in excess
- Frequency of feeding: three times daily
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
91 d
Hardness:
54 ± 6.4 mg/L as CaCO3
Test temperature:
10.7 ± 0.7 °C
pH:
6.97 ± 0.39
Dissolved oxygen:
9.4 ± 0.7 mg/L
Nominal and measured concentrations:
Measured: 6.0, 10.3, 23.1, 53.0 and 114 µg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups: oscillating incubation chambers of 100 mL beakers with bottoms removed and replaced with nylon mesh
- Test vessel: 2 L exposure chambers
- Type of flow-through: diluter
- No. of fertilized eggs/embryos per vessel:100
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water obtained from municipal water supply of lake Superior , WI, USA. The water was dechlorinated using an activated charcoal filter and passed through a cation exchange resin to remove heavy metals. Sodium sulfite was then added to remove any residual chloramines.
- Alkalinity:41.0 ± 3 mg/L
- Conductivity: 95 µmhos/cm
- Intervals of water quality measurement: Temperature was measured daily. Dissolved oxygen was measured ever 48 hrs. Hardness of water, alkalinity, pH and conductivity were measured weekly throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16h light/ 8h dark

EFFECT PARAMETERS MEASURED: Egg hatchability, survival and growth of rainbow trout was recorded after 91d of exposure.

POST-HATCH DETAILS
- Begin of post-hatch period: day 29-39 days
- No. of hatched eggs (alevins)/treatment released to the test chamber: 15 fry per test chamber
- Release of alevins from incubation cups to test chamber on day no.: approx day 45
Reference substance (positive control):
no
Duration:
91 d
Dose descriptor:
NOEC
Effect conc.:
6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth as measured by mean standard length, wet weight and dry weight of larvae
Duration:
91 d
Dose descriptor:
LOEC
Effect conc.:
10.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth as measured by mean standard length, wet weight and dry weight of larvae
Duration:
91 d
Dose descriptor:
other: EC20
Effect conc.:
8.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth as measured by mean standard length, wet weight and dry weight of larvae
Details on results:
Hatching of rainbow trout eggs occurred approximately 34 d after fertilization and hatching success was not significantly affected at any
of the tested concentrations. Mortality of larvae in the two highest concentrations of 53 and 114 µg/L was 95.5 and 100%, respectively, by 3 d posthatch. No larvae survived to the end of the test at these two concentrations. Mortality began by 8 d posthatch in the third highest concentration of 23.1 µg/L and approximately 67% of the fish died by the end of the test. Mortality was not significantly different from the control at lower concentrations with 90% of the larvae surviving to the end of the test. Growth, as measured by mean standard length, wet weight, and dry weight of larvae at test termination, was significantly less than the control at 10.3 µg/L. Exposure to 23.1 µg/L resulted in greater growth decreases from the
control. No significant effects were observed at the lowest concentration of 6.0 µg/L. The NOEC, LOEC, and chronic value for rainbow trout based nonylphenol effects on growth after 91 d were 6.0, 10.3, and 7.9 mg/L, respectively.
Validity criteria fulfilled:
not specified
Executive summary:

The 91-day long-term toxicity of nonylphenol to early life stage of rainbow trout (Oncorhynchus mykiss) was studied under flow through conditions. 100 fertilized eggs per test concentration of Oncorhynchus mykiss were exposed to measured concentrations of 6.0, 10.3, 23.1, 53.0 and 114 µg/L nonylphenol. The test system was maintained at ~ 11 ºC and a pH of 6.97. Mean percent hatch of any test concentration was not significantly different from controls. Time to hatch was 39±5 days with swim-up at approximately at day 45. The most sensitive end point was growth. The 91-day NOEC value, based on sublethal effects (growth measured by mean standard length, wet weight and dry weight) is determined to be 6.0 µg/L.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
adopted in 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 19.04.2017
- Expiration date of the lot/batch: 2022-04-19
- Purity: 100% (UVCB)
Analytical monitoring:
yes
Details on sampling:
Samples of test media including solvent control group and control group were taken from alternating test replicates on days -2, -1 and during the exposure on study days 0, 6, 13, 20, 27 and 33. Additional analysis of the nominal concentration 25.0 µg/L was carried out from replicate 2 on study day 1 due to high egg mortality in replicate 2 on day 1. Additional analysis of all replicates of the solvent control and replicate 2 of the nominal concentration 100 µg/L was carried out on study day 34 due to analytical findings in replicate 2 of the solvent control and unexpected recovery values in the nominal concentration 100 µg/L. The stock solution was sampled and analyzed from freshly prepared and corresponding 4 days aged stock solution of one application interval on study days 9 and 13.
At each sampling day 2 samples were taken per (alternating) test replicate. For each sample 100 mL of each test item concentration, the solvent control and the control were sampled and stabilized with 100 mL isopropanol. For analysis of fresh and aged stock solutions, approx. 10 mL of each stock solution and the solvent were sampled.

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 1000 mg/L was prepared in methanol. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the solvent into a glass flask. The solution was agitated until it was visually clear. Further stock solutions (0.5 g/L, 0.25 g/L, 0.125 g/L, 0.0625 g/L) were prepared by dilution with methanol. The stock solutions were prepared in appropriate intervals of 3 to 4 days. Syringes were filled with the freshly prepared stock solutions or pure methanol for the solvent control every 3 to 4 days.
Precision syringe pumps were used for the introduction of stock solutions of the test item dissolved in methanol into the mixing chamber. The stock solutions and the respective amount of dilution water to form the test concentrations were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.05 mL/L
- Other relevant information: An equilibration period of 22 days was carried out prior to start of the exposure and monitored analytically at 8 time points. As the recoveries were not in the expected range in the first measurements, equilibration was continued until the measured concentrations were approx. within the range of ± 20% of the nominal concentration.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (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
- Method of collection of fertilised eggs: 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 (less than 30%).
- Subsequent handling of eggs: About 1000 eggs were taken and washed in dilution water.

POST-HATCH FEEDING
- Start date: The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 50 eggs, resulting in a total of 100 eggs per test concentration).
- Feeding: The feeding regime was ad libitum during the whole feeding period (study day 5 to 34). Feeding started 3 days after the beginning of hatch (on study day 5 (post-hatch day 1)). Larvae were fed with a starter food (AQUA SCHWARZ GMBH, Göttingen, Germany), as well as a suspension of the starter food and fine milled brine shrimp nauplii (2 – 5 times daily). About 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2 – 6 times daily).
Brine shrimp nauplii origin, breeding conditions: Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, 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.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d
Hardness:
62 - 82 mg CaCO3/L
Test temperature:
25.8 - 26.6 °C
pH:
7.06 - 7.89
Dissolved oxygen:
67 - 99% saturation
Salinity:
-
Conductivity:
146 µS/cm
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25, 50 and 100 µg/L
Measured (TWA): 4.27, 10.9, 21.3, 56.2 and 94.4 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.
- Aeration: The dilution water supply tank was aerated. No additional aeration of the test vessels was provided.
- Type of flow-through (e.g. peristaltic or proportional diluter): Membrane piston pumps provided the water flow. Precision syringe pumps were used for the introduction of stock solutions of the test item dissolved in methanol into the mixing chamber. The stock solutions and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L) where the eggs/fish were exposed. One mixing chamber was used for one test aquariuma (replicate). The accuracy of the water flow was checked prior to start of the exposure and three times per week thereafter. Water exchange in the test aquaria was about 5 test vessel volumes per day (1.563 L/h).
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 80
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: 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.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
- Alkalinity: 0.6 mmol/L
- Total organic carbon: The total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 2, 7, 14, 21 and 28. Mean TOC was 1.39 mg/L throughout exposure.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured in dilution water and one control vessel once per hour. Dissolved oxygen was measured in all replicates and flow rates were determined 3 times per week. Temperature and pH in all replicates, TOC and chlorine from dilution water and total hardness were measured weekly. The light intensity on the surface of the test aquaria was measured at the start of the exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light: 8h dark
- Light intensity: 300 ± 150 Lux

EFFECT PARAMETERS MEASURED: The number of hatched larvae was determined daily until study day 7. Egg, embryo, larvae and juvenile fish mortality was recorded. 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. quiescence, hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate. Body length and body weight of all survivors were measured.

VEHICLE CONTROL PERFORMED: yes

POST-HATCH DETAILS
- Begin of post-hatch period: On study day 4, 91% of the control and 93 % the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).

FERTILIZATION SUCCESS STUDY
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 discarded. The fertilization rate was 94%.
Reference substance (positive control):
no
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
4.27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
13.9 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
56.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other:
Basis for effect:
length
Remarks:
fry growth
Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
69.1 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
length
Remarks:
fry growth
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
10.9 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: 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 80% in the control and 82% in the solvent control. A concentration-related decrease of the post-hatch survival was detected with increasing test concentrations.
The cumulative survival at the end of the exposure, related to the number of eggs introduced on day 0 was 75% in the control group and 76% in the solvent control group. This clearly exceeded the validity criteria of the guideline given in section 9. A concentration related decrease of the overall survival in the highest three test concentrations was detected with increasing test concentrations.
- Days to hatch or time to release of young: Hatch began on study day 3 in the control and on day 2 in the solvent control and all test item concentrations and continued until study day 5. The hatch of the control, solvent control and all test item concentrations was completed until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 91% in the control and 93% in the control solvent control.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): Swim-up was observed for a 3-day period from study days 4 to 6. Newly hatched fry began to swim up on study day 5 (PHD 1). On study day 6 (PHD 2), all surviving larvae had swum up. No statistical analysis of swim-up data was carried out.
- Incidents in the course of the test which might have influenced the results: On study day 30, the stock solution source for solvent control replicate 2 and the nominal concentration 6.25 µg/L replicate 3 were accidentally swapped. Therefore, the concentration in replicate 3 of the lowest concentration could potentially have decreased in the last four days of the study. However, no effects in any replicate of this concentration occurred between study day 30 and 34. Additionally, the statistical effect levels are identical in case replicate 3 is removed from the statistical analysis. Therefore, we consider it justifiable to perform the statistical analysis with all test replicates.
- Type of and number with morphological/behavioral abnormalities: No biologically significant morphological and behavioral effects were observed in the control, the solvent control and the test item concentrations of 4.27 and 10.9 µg/L. In the three highest test concentrations biologically significant behavioral effects as quiescence (Q), lethargy (L), missing tail fin (F), malformed tail fin (M), altered swimming behavior (B), stuck position (P), side position (S), paleness (Pl) and tumbling (T) were observed from study day 6 up to study day 34.
Validity criteria fulfilled:
yes
Conclusions:
In a GLP study according to OECD guideline 210 (Fish Early life stage test), significant effects on early life stages could be observed with hatching success being the most sensitive endpoint (34d-NOEC = 4.27 µg/L). Hence, the test substance is considered to be very toxic to fish based on long-term exposure.
Endpoint:
fish early-life stage toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see Analogue justification document in chapter 13
Reason / purpose:
read-across source
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
4.27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
number hatched

Description of key information

NOEC (33d) = 0.00427 mg/L (measured TWA) for hatching success of Danio rerio (OECD 210, read across)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.004 mg/L

Additional information

Since no study assessing the long-term toxicity of Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched (CAS 244235-47-0) to fish is available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 Grouping of substances, a read-across to the structurally very similar source substance Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched (CAS 174333-80-3), was conducted. The only structural difference between the source substance and the main component of the target substance is the lack of a methyl group at the oxime carbon of the source substance. The read across is justified due to (i) the similarity of structure and functional groups and accordingly (ii) similar physico-chemical properties resulting in a similar environmental fate and ecotoxicity profile (see table below).

Substance

Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched

Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched

CAS number

244235-47-0

174333-80-3

Structure

see attachment

see attachment

Molecular formula

C17H27NO2

C16O2NH25

Molecular weight

~ 277 g/mole

~ 263 g/mole

PC parameter

 

 

Water solubility

 > 0.02 < 0.1 mg/L (EU method A.6)

0.4 mg/L (EU method A.6)

Partition coefficient

> 5.7 (EU method A.8)

5.5 (EU method A.8)

Vapour pressure

< 1.5 Pa at 20 °C (OECD 104)

0.37 Pa at 20 °C (OECD 104)

Environmental fate

 

 

Biodegradability

1 % in 28 days (BODIS)

0 % in 28 days (OECD 302c)

Adsorption [log KOC]

3.9 (OECD 121)

3.7 (OECD 121)

Hydrolysis

not relevant due to very low water solubility

Ecotoxicology

 

 

Short-term toxicity to fish

[96h-LC50]

0.46 mg/L (EU method C.1)

1.1 mg/L (EU method C.1)

Short-term toxicity to aquatic invertebrates

[48h-EC50]

-

2.7 mg/L (EU method C.2)

Long-term toxicity to aquatic invertebrates

[21d-NOEC]

2.8 mg/L (OECD 211)

0.189 mg/L (OECD 211)

Short-term toxicity to algae

[72h-EC50]

760 mg/L (OECD 201)

36.3 mg/L (OECD 201)

Long-term toxicity to algae

[72h-NOEC]

472 mg/L (OECD 201)

14.9 mg/L (OECD 201)

Toxicity to microorganisms

[3h-EC10]

260.1mg/L (OECD 209)

200.4 mg/L (OECD 209)

The key study with the source substance was performed according to GLP and OECD guideline 210 using Danio rerio as test organism (Herrmann 2019). The test substance showed significant effects 30 days post hatch when tested with time-weighted arithmetic mean measured concentrations ≥4.27 µg/L. The parameter hatch showed to be the most sensitive endpoint with a NOEC of 4.27 µg/L. For the parameters post-hatch survival and overall survival (mortality) the NOEC was 10.9 µg/L and for the parameter fry growth (expressed as length and fresh weight) 56.2 µg/L. All effect values are given based on the time-weighted arithmetic mean measured values of the peak groups B and C of the test item. Based on this result the substance is considered to be very toxic to fish at chronic exposure.

A supporting study with the minor constituent 4-nonylphenol to early life stage of rainbow trout (Oncorhynchus mykiss) under flow through conditions (Spehar et al. 2010) confirmed this conclusion. The study was performed according to ASTM guideline E 1241-05 over an exposure time of 91 days. 100 fertilized eggs per test concentration were exposed to measured concentrations of 6.0, 10.3, 23.1, 53.0 and 114 µg/L 4-nonylphenol. The test system was maintained at ~ 11 ºC and a pH of 6.97. Mean percent hatch of any test concentration was not significantly different from controls. Time to hatch was 39 ± 5 days with swim-up at approximately at day 45. The most sensitive end point was growth. The 91-day NOEC value, based on sublethal effects (growth measured by mean standard length, wet weight and dry weight) is determined to be 6.0 µg/L.

Based on the reasons given above the NOEC value of 0.00427 mg/L is considered to be sufficiently conservative to be used for PNEC derivation of Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched (CAS 244235-47-0).