1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The stability ot test concentration during exposure was examined by chemical analysis (HPLC) at 0, 24, 48 and 72 hours.
Aliquots of the samples from the biological test were directly analysed by HPLC/MS/MS. Samples were diluted with Acetonitrile (1:1; v/v). The samples were taken on 2017-09-12, on 2017-09-14 and on 2017-09-15 and were stored deep frozen at - 18 °C and were analysed on 2017-10-02.

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations:
- A stock solution was prepared to give the desired series of test concentrations
- 200.0 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer
- Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 μm
- The pH was measured to be 7.8.
- Appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 1.667 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL
- For each test item concentration and the control 3 replicates were prepared
- All flasks were sealed with cotton stoppers

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre-cultures : Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growtht medium.
- Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

23 +/- 2 °C

pH:

7.9 - 10.2

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 25, 50 and 100 mg/L; Effective concentrations ranged from 0.32 % to 1.1 % of nominal values at 0 hours, from 0.46 % to 0.62 % of nominal values at 24 hours , from 0.37 % to 0.60 % of nominal values at 48 hours and from 0.42 % to 0.82 % of nominal values at 72 hours.

Details on test conditions:

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
- Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, was used. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design: 3 test concentration plus 1 control, 3 replicates per concentration, 3 replicates per control, Initial cell density in the test cultures approximately 5000 cells per millilitre, Additionally highest test concentration without algae
- Test item concentration: 25, 50 and 100 mg/L (nominal)
- Method of administration: stock solution
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.45 (mg/L)

Remarks:

Analytical (arithmetic mean measured)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.45 mg/L

Remarks:

Analytical (arithmetic mean measured)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: ≥ 0.45 mg/L

Remarks:

Analytical (arithmetic mean measured)

Details on results:

The results are expressed in terms of nominal concentrations and arithmetic mean measured concentrations. Effective concentrations ranged from 0.32 % to 1.1 % of nominal values at 0 hours, from 0.46 % to 0.62 % of nominal values at 24 hours , from 0.37 % to 0.60 % of nominal values at 48 hours and from 0.42 % to 0.82 % of nominal values at 72 hours.

Validity criteria fulfilled:

yes

Remarks:

the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16; the mean coefficient of variation must not be higher than 35%; the coefficient of variation of the mean specific growth rate, must not exceed 7%

Conclusions:

No toxic effects against algae were observed at the limit of water solubility (< 3µg/L) under exposure conditions. For details see chapter 4.8. The results are expressed in terms of nominal concentrations and arithmetic mean measured concentrations. Effective concentrations ranged from 0.32 % to 1.1 % of nominal values at 0 hours, from 0.46 % to 0.62 % of nominal values at 24 hours , from 0.37 % to 0.60 % of nominal values at 48 hours and from 0.42 % to 0.82 % of nominal values at 72 hours.

Executive summary:

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 1 mg/L of Solvent Green 28 dissolved in water. In the main test, the algae were exposed to the test item added to dilution water at a range of concentrations for a period of 72 hours. Defined concentrations of the test item led to a certain inhibition of algal yield and growth rate at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. All calculations were carried out using the statistics program ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000. During the test a temperature range of 21 - 24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure. The maintenance of the test item concentrations was proved by analytical measurements. In order to avoid an impairment of the test system, an additional replicate for each test item concentration was used for analysis at all measuring points (0, 24, 48 and 72 hours) and for pH measurement at the beginning of the test. PH measurement at the end of the test were performed using replicate I of the test concentrations and the control vessels. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, at all measuring points (0, 24, 48 and 72 hours) a test flask without algae was run in parallel to the geometric series of test concentrations. The validity criteria of the test were met: The cell density in the control cultures increased by a factor of at least 16 within 72 hours. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %. No toxic effects against algae were observed at the limit of water solubility (< 3µg/L) under exposure conditions. For details see chapter 4.8. The results are expressed in terms of nominal concentrations and arithmetic mean measured concentrations. Effective concentrations ranged from 0.32 % to 1.1 % of nominal values at 0 hours, from 0.46 % to 0.62 % of nominal values at 24 hours, from 0.37 % to 0.60 % of nominal values at 48 hours and from 0.42 % to 0.82 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the algae study.

Description of key information

72h-ErC10 and ErC50 (Desmodesmus subspicatus) > solubility limit; OECD TG 201; Spoo-Kloeppel (2018).

Additional information

Experimental algae studies are available on other substances of the category: Reinblau RLW (CAS No 41611-76-1) and Solvent Violet 36 (CAS No 82-16-6), with a similar result. See the category approach justification document, attached in section 13, for further details.

Global overview of the category approach for the endpoint:

	Solvent Violet 36	Solvent Green 3	Reinblau RLW	Reinblau BLW	Solvent Blue 104^#	Solvent Green 28
Toxicity to aquatic algae	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal) or > 0.16 mg/L (measured)^$   72h-NOECr and ErC50 (algae) > solubility limit*	72h-NOECr and ErC50 (algae) > solubility limit*   Other Lead Registrant data available but not considered key	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal)^$   72h-NOECr and ErC50 (algae) > solubility limit*	Study planned (OECD TG 201)   72h-NOECr and ErC50 (algae) > solubility limit*	72h-NOECr and ErC50 (algae) > solubility limit*	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal)^$

^# not registered by LANXESS

* iSafeRat® High Accuracy QSAR predictions (KREATiS, 2022)

^$ experimental studies