Reaction mass of 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Sep 2020 - 03 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Analytical samples were taken from all replicates at all test item concentrations and controls at day 3 of the equilibration phase, at test start (day 0), days 7, 14, 21, 28 and at test end (day 35). At each sampling also a retain sample was taken.
- 10 mL samples were taken and stabilized with 10 mL acetonitrile.
- Additional samples were taken from all treatments at day 22 after a malfunction of the dosing system.
Additionally 1mL samples were taken from fresh and aged stock solutions at every stock solution renewal. These samples were not additionally stabilized.
- All samples were stored deep frozen until they were transferred to the analytical laboratory.
- Samples analyzed: Analytical samples taken from the negative control, solvent control and all test item concentrations were analyzed in duplicate. At day 14 and 35 analyses were performed of one replicate of both controls. At day 7, 14, 21, 28 and 35 analyses were performed at all concentration levels changing systematically amongst the replicates. Additional analysis was performed in the test concertation of 1.10 µg/L at day 22 after a short malfunctioning of the dosing system.

Vehicle:

yes

Remarks:

Solvent: DMF

Details on test solutions:

The quantities of test item required for preparing all stock solutions were weighed on a weighing scoop and transferred to a volumetric flask. The stock solutions were prepared with 100 mL DMF by mixing and shaking thoroughly until the test item was dissolved. Lower concentration stock solutions were prepared by dilution of the higher concentrated stock solutions. Stock solutions and dilutions were prepared at test start (introduction of fertilised eggs) and thereafter every 7 days. Stock solutions/dilutions had a yellowish staining decreasing in intensity with decreasing concentration. The stability of the test item in the stock solutions during the renewal period was confirmed in a non-GLP pre-experiment. The stock solutions/dilutions were stored in 100 mL glass bottles, shielded against light. For each concentration 0.05 mL of the appropriate stock solution was taken from these bottles with a syringe pump and mixed in a mixing chamber with 1 L test medium applied by a time-controlled valve. From the mixing chamber the 1 L test solution was transferred to one of the four replicates of each treatment by a computer controlled consecutive valve cascade. Prior to exposure start an equilibration phase of the dosing/exposure system of 7 days was performed.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM:
Zebrafish Danio rerio Hamilton (Cypriniformes: Cyprinidae) was used as the test organism. A brood strain was continuously reared in the in-house laboratory. This strain, originally bred from wildstrain Zebrafish, was originally purchased in a healthy condition from Fraunhofer IME (Schmallenberg, Germany).

CULTURING:
- The fish were cultured in groups of approximately of 100 – 400 fish in ISO test water.
- The fish were cultured at a temperature of 25 ± 2 °C in a climate-controlled room with 16 hours of illumination and 8 hours of darkness.
- The animals were fed with commercial fish flake food (TetraMin, Tetra GmbH, Germany) and protein-enriched pellet food (Caviar, BernAqua, Belgium) at least once per day.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS:
- Numbers of parental fish: group spawning.
- Method of collection of fertilised eggs: trays were placed in the culturing tanks directly after turning on the light in the morning. After approximately 2.5 hours of spawning, the spawning trays were removed and eggs were collected.

POST-HATCH FEEDING:
- Newly hatched larvae: a suspension of artificial fry food consisting of commercial fry dust food (Caviar 50-100 µm, BernAqua, Belgium) and rotifers (AquaHobby Fischfutterhandel, Germany).
- Fry: a suspension of commercial fry dust food (Caviar 50-100 and 100-200 µm, BernAqua, Belgium) and rotifers (AquaHobby Fischfutterhandel, Germany).
- This was superseded by a suspension of commercial fry food (Caviar 100-200 µm, BernAqua, Belgium) and ground commercial fish food (TetraMin, Tetra GmbH, Germany).
- Feeds were provided 3 - 7 times a day.
- In addition to this, newly hatched brine shrimp nauplii were fed ad libitum.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Hardness:

13-14 °dH, corresponding to 232-250 mg/L CaCO3.

Test temperature:

Temperature in vessels: 25.1 - 26.6 °C
Temperature continuously measured: 24.8 - 27.0°C

pH:

7.30 - 7.62

Dissolved oxygen:

68 - 100 % Oxygen concentration

Nominal and measured concentrations:

Nominal concentrations: Control, Solvent Control, 1.1, 3.48, 11.0, 34.8 and 110 µg/L.
Measured concentration: The measured content of the test item was between 72 and 118 % of nominal. Since the arithmetic mean measured content of the test item in the samples was between 80 and 120 % of nominal (86 - 97 %) the toxicological endpoints were evaluated using nominal concentrations. For detailed results of the measured concentration, see "Any other information on results".

Details on test conditions:

TEST SYSTEM:
- Vessels: 14 L glass chambers filled with 8 L test solution.
- Breeding cages: Glass cylinders closed with a stainless steel mesh at the bottom.
- Aeration: none
- Type of flow-through: peristaltic pump
- Renewal rate of test solution: 6 test chamber volumes per 24 h.
- No. of fertilized eggs/embryos per vessel: 20 embryos per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: The loading rate (biomass per volume of test solution) did not exceed 0.5 g fish (wet weight)/L at any time.

TEST MEDIUM / WATER PARAMETERS:
The test medium was reconstituted test water (ISO water; OECD test guideline 203 (2019)) consisting of analytical grade salts dissolved in purified water with a conductivity of 711 µS/cm. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity.
At exposure initiation the mean pH-value of the untreated test medium (measured in the control replicates) was 7.56, the dissolved oxygen concentration was 100 % of the air saturation and the total hardness was 14°dH (250 mg/L as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of illumination and 8 hours of darkness.

EFFECT PARAMETERS MEASURED :
- Hatching and mortality of the fish were observed daily.
- Observations of sublethal effects and behavioral abnormalities were performed daily.
- The weight and total length of each fish were determined at the end of the test. Immediately after euthanization the total length and wet (bottled dry) were measure individually. For determination of dry weight all fish from one replicate were pooled together, died at 60 C for 120h and overall dry weight was measured.

VEHICLE CONTROL PERFORMED: yes

POST-HATCH DETAILS:
- Begin of post-hatch period: All fish hatched within day 3 to 6 after test start.
- Release from the incubation cage to the test chamber: The fish hatched inside the incubation cages and were released to the test aquaria on test day 13.

FERTILIZATION SUCCESS STUDY:
- Hatching success at the negative control: 100 %
- Hatching success at the solvent control: 98.8%
- Hatching success at the pooled control: 99.4%

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

70.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% CI: 59.6 - 82.6

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

69.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 64.6 - 74.7 µg/L

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

69.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: 95% CI: 64.6 - 74.6 µg/L

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

15.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Fresh weight

Remarks on result:

other: 95% Cl: 6.92- 24.4 µg/L

Key result

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

> 110 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Fresh and dry weight

Key result

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

> 110 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

86.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Cumulative mortality

Remarks on result:

other: 95% CL: 80.1 - 93.2 µg/L

Key result

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

86.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Remarks on result:

other: 95% CL: 80.0 - 93.2 µg/L

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Fresh and Dry weight

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

34.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Cumulative mortality

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

34.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

110 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

HATCHING SUCCES AND MORTALITY:
At all concentrations, no statistically significant effect on hatching success was observed compared to the pooled controls. The mean hatching success in the controls and the treatment groups ranged from 97.5 to 100 % without concentration-effect relationship.

OBSERVATIONS:
- At the concentration levels up to and including 34.8 µg/L single fish of smaller size were recorded.
- At 110 µg/L fish showed partly delayed development (fish were visibly smaller).
- Starting test day 20, fish developed a slight orange body staining at 34.8 µg/L and a noticeable orange body staining at 110 µg/L.

BODY LENGHT AND WEIGHT:
On fresh weight, dry weight and total body length of fish at test end, no statistically significant influence could be detected at the concentrations up to and including 11.0 µg/L compared to the pooled controls. At 34.8 and 110 µg/L a statistically significant reduction was detected compared to the pooled controls.

ANALYTICAL RESULTS:
The measured content of the test item was between 72 and 118 % of nominal. Since the arithmetic mean measured content of the test item in the samples was between 80 and 120 % of nominal (86 - 97 %) the toxicological endpoints were evaluated using nominal concentrations. For detailed results of the measured concentration, see "Any other information on results"

Reported statistics and error estimates:

COMPARISON OF NEGATIVE CONTROL VS. SOLVENT CONTROL :
There was no statistical difference between the negative control and the solvent control for endpoints hatching success, total length, fresh and dry weight. Following the guidance for NOEC determination in OECD-guideline 210, Appendix 5 (OECD 2013), all statistical evaluations were compared to the pooled controls. For end-points post-hatch survival and cumulative mortality, a statistical significant difference between the negative and the solvent control was observed. In such a case, data is usually compared to the solvent control only. Because of the increased mortality in the solvent control compared to the negative control and the three lowest test concentrations (1.10, 3.48, and 11.0 µg/L) and in order to achieve a statistically significant and reliable concentration-response, end-points were compared to the pooled controls nevertheless.

HATCHING SUCCES:
- NOEC/LOEC: normality check and a Levene’s Test for Variance Homogeneity (α = 0.01). Since normal-distribution requirements were fulfilled, but variance homogeneity check failed, the Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was chosen.
- EC VALUES: Weibull analysis with linear maximum likelihood regression and control compensation (Abbot’s formula). Significance level was α = 0.05, one-sided smaller; confidence limits based on Fieller’s theorem

POST HATCH SURVIVAL AND CUMULATIVE MORTALITY
- NOEC/LOEC: normality check and a Levene’s Test for Variance Homogeneity (α = 0.01).
Since normal-distribution variance homogeneity requirements were fulfilled the Williams multiple t-test was performed (α = 0.05, one-sided smaller).
- EC VALUES: Logit analysis with linear maximum likelihood regression and control compensation (Abbot’s formula). Significance level was α = 0.05, one-sided smaller; confidence limits based on Fieller’s theorem.
All statistical calculations were carried out using the software-program ToxRat 3.3.0.

Table: Hatching, mortality, mean length and body weight of test organisms at termination of the test (day 35)

2- Amylanthraquinone [µg/L]	Hatching Success (day 6) [%]	Post hatch survival [%]	Cumulative mortality [%]	Mean fish length [mm]	Mean fish fresh weight [mg]	Mean fish dry weight [mg]
Negative Control	100.0	98.8	1.3	22.3	102.6	28.6
Solvent Control	98.8	89.9	11.3	22.1	103.7	29.0
1.10	100.0	96.3	3.8	21.9	98.9	27.9
3.48	100.0	96.3	3.8	22.0	98.8	28.0
11.0	97.5	96.2	6.3	21.6	94.9	27.6
34.8	100.0	96.3	3.8	20.9	87.0	24.7
110	100.0	7.5	92.5	19.1	65.3	16.2

Table: ECx, NOEC and LOEC-values after 35 days of exposure to the test item (based on nominal concentrations in µg/L). 

Endpoint	Hatching Success	Post Hatch survival	Cummulative mortality	Length	Fresh weight	Dry Weight
NOEC	110	34.8	34.8	11.0	11.0	11.0
LOEC	>110	110	110	34.8	34.8	34.8
EC10		69.3 (64.6-74.6)	69.4 (64.6-74.7)	70.9 (59.6-82.6)	15.7 (6.92-24.4)	23.5 (12.3-33.4)
EC20		75.2 (69.9-81.0)	75.2 (69.9-81)	>110	43.2 (29.2-56.0)	46.6 (32.7-57.6)
EC50		86.2 (80.0-83.2)	86.3 (80.1-93.2)	>110	>110	>110

( ) between the brackets the 95% confidence limits are shown.

 

Table:MEasured concentrations of 2-Amylanthraquinone in test solution during the test.

Nominal test concentration (µg/L)	Day 0 (µg/L)	Day 7 (µg/L)	Day 14 (µg/L)	Day 21 (µg/L)	Day 28 (µg/L)	Day 35 (µg/L)	Arithmic mean (% of normal)
Negative Control	n.d	-	n.d.	-	-	n.d.	-
Solvent control	n.d.	-	n.d.	-	-	n.d.	-
1.10	0.740 0.888	0.844	0.866	0.968	0.728	0.968	86
3.48	3.80 3.22	3.12	3.30	2.70	2.32	2.66	91
11.0	10.8 11.3	9.52	10.0	9.16	7.48	8.16	92
34.8	33.0 31.8	33.2	33.0	29.8	24.6	28.4	94
110	101  104	99	100	95.0	91.5	99.5	97

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks"

Conclusions:

According to the results of the test, the overall NOEC (35 d) was determined to be 11.0 µg/L
2-Amylanthraquinone (nominal). The overall LOEC (35 days) was determined to be 34.8 µg/L 2-Amylanthraquinone (nominal). The EC10 and EC20 (35 days) value of the most sensitive parameter (fresh weight) were determined to be 15.7 µg/L and 43.2 µg/L 2-Amylanthraquinone (nominal). The EC50 (35 days) value of the most sensitive parameter (post-hatch survival) was determined to be 86.2 µg/L 2-Amylanthraquinone (nominal).

Executive summary:

An early life stage test was 2-amylanthraquinone was performed according to OECD guideline 210 and GLP guidelines under flow through conditions. The study started with fertilized Danio Rerio eggs, exposed to a concentration of 1.10, 3.48, 11.0, 34.8 and 110  µg/L for 35 days. Analytical samples taken from the negative control, solvent control and all test item concentrations were analyzed in duplicate. At day 14 and 35 analyses were performed of one replicate of both controls. At day 7, 14, 21, 28 and 35 analyses were performed at all concentration levels changing systematically amongst the replicates. The arithmic mean measured content of test item was between 86 and 97% of nominal, therefore the toxicological endpoints were evaluated using the nominal concentrations. According to the results of the test, the overall NOEC (35 d) was determined to be 11.0 µg/L 2-Amylanthraquinone (nominal). The overall LOEC (35 days) was determined to be 34.8 µg/L 2-Amylanthraquinone (nominal). The EC10 and EC20 (35 days) value of the most sensitive parameter (fresh weight) were determined to be 15.7 µg/L and 43.2 µg/L 2-Amylanthraquinone (nominal). The EC50 (35 days) values of the most sensitive parameter (post-hatch survival) was determined to be 86.2 µg/L 2-Amylanthraquinone (nominal). The study fulfilled the validity criteria and is considered reliable without restrictions.

Description of key information

An early life stage test was 2-amylantraquine was performed according to OECD guideline 210 and GLP guidelines under flow through conditions. The study started with fertilized Danio Rerio eggs, exposed to a concentration of 1.10, 3.48, 11.0, 34.8 and 110 µg/L for 35 days. Analytical samples taken from the negative control, solvent control and all test item concentrations were analyzed in duplicate. At day 14 and 35 analyses were performed of one replicate of both controls. At day 7, 14, 21, 28 and 35 analyses were performed at all concentration levels changing systematically amongst the replicates. The arithmic mean measured content of test item was between 86 and 97% of nominal, therefore the toxicological endpoints were evaluated using the nominal concentrations. According to the results of the test, the overall NOEC (35 d) was determined to be 11.0 µg/L 2-Amylanthraquinone (nominal). The overall LOEC (35 days) was determined to be 34.8 µg/L 2-Amylanthraquinone (nominal). The EC10 and EC20 (35 days) value of the most sensitive parameter (fresh weight) were determined to be 15.7 µg/L and 43.2 µg/L 2-Amylanthraquinone (nominal). The EC50 (35 days) values of the most sensitive parameter (post-hatch survival) was determined to be 86.2 µg/L 2-Amylanthraquinone (nominal). The study fulfilled the validity criteria and is considered reliable without restrictions.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

15.7 µg/L