1,4-dichloro-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

Remarks:

High Performance Liquid Chromatography coupled to a tandem Mass Spectrometry (HPLC-MS/MS)

Details on sampling:

Sampling for Chemical Analysis:
- Samples of freshly prepared test media were taken from all five test solutions and the control at the beginning of the test
- Samples of aged test concentrations were taken at the end of the test (48 hours) directly from pooled replicates per test concentration and control

Preparation and Storage:
- A volume of 7.5 mL of sample was used and all samples were stabilized with 7.5 mL of acetonitrile immediately after the sampling
- Duplicate samples from all test levels and untreated controls were stored at -20°C in a refrigerator and protected rom light until shipment for analysis
- For security reasons, a reserve set of stabilized samples was stored deep frozen

Vehicle:

no

Details on test solutions:

Due to the very low water solubility of the test item (0.0118 mg/L at 20°C) a Water Accommodated Fraction (WAF) of the test item was prepared according to the OECD guidance No. 23.

PREPARATION OF THE TEST MEDIA:
- 128.9 mg of the test item were transferred into a 1 L sterile glass flask (acetone washed and sterilised)
- Sterile growth medium (1 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottle and stirred for 72 h at about 20°C followed by 30 minutes ultrasonic treatment
- The WAFs were filtered (0.22 μm PES filter, polyethylensulfone, Nalgene, bottle top filter, sterile conditions); after filtration the test solution appeared to be slightly reddish
- This test solution was diluted with sterile growth medium to obtain the other test concentrations
- All work was conducted under a clean bench using sterile equipment

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
- Cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980); prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially growing algae for the test
- Age: The culture duration of the pre-cultures was 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21.8-22.0 °C

pH:

7.62 - 7.83

Dissolved oxygen:

n.a.

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

- At test start the measured concentrations were 0.041, 0.080, 0.157, 0.332 and 0.568 mg test item/L (0.57–0.66 % of nominal)
- After 72-hours, the measured test item concentrations were 0.001, 0.004, 0.044, 0.135, 0.339 mg test item/L (0.02–0.34 % of nominal; 2.76–59.59 % of initial)
- The measured concentration at test initiation were not between 80–120 % of nominal and at test initiation the test item concentration decreased by more than 20 %
- Therefore, the evaluation of the test was based on the geometric mean measured concentrations of 0.007, 0.019, 0.083, 0.212 and 0.439 mg test item/L.

Details on test conditions:

EXPOSURE CONDITIONS:
- Test vessels: conical glass flasks (250 mL) covered with air-permeable silicone-sponge caps (sterilized)
- Volume of test solution per test vessel: 100±5 mL
- Age of the pre-culture: 4 days
- Number of cells per mL in the pre-culture before inoculating the test solutions: 95.5x10E+04
- Number of cells per mL test solution at the beginning of the test: 0.3x10E+04
- Number of replicates per test item concentration: 4
- Number of replicates in the control: 6
- Number of replicates for stability check (highest concentration level without algae): 1 (without algae)
- Test concentration: 5 (6.25, 12.5, 25.0, 50.0 und 100 mg test item/L)
- Test medium: OECD medium
- pH in test solutions: 7.6-7.8
- Type of light: Econlux LED Sunstrip "daylight"
- Light intensity: 60-120 μE m-2 s-1 (4440 - 8880 lux)
- Temperature: 21-24 °C, controlled at +/- 2 °C
- Shaker: oscillated by continuously stirring with 150 rpm
- Counting of algae: Daily

WATER QUALITY MEASUREMENTS:
- pH-value and temperature was measured and recorded daily in the test concentrations and control

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- The geometric mean measured concentrations of 0.44 mg test item/L corresponds to a nominal loading of 100 mg/L.
- The NOEC for growth rate was determined to be at 0.08 mg/L based on geometric mean measured test item concentrations. However, due to the inhibition of 4.68 % at 0.21 mg/L and 2.99 at 0.44 mg/L, the NOEC was set to be above the highest test concentration of 0.44 mg/L, equivalent to a nominal loading of 100 mg test item/L, by expert judgement. The LOEC was set to be above 0.44 mg/L. Effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant. Generally, it is recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (December 2017) ErC50 value of 2.90 mg/L (nominal; 95% confidence limits: 2.66 – 3.16 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L

Reported statistics and error estimates:

Since no inhibition above 50% was observed, results were not statistically analysed to determine EC50 and EC10 values with 95 % confidence intervals.

Validity criteria fulfilled:

yes

Remarks:

The cell number in the control cultures increased by a factor of 82.6; the mean coefficient of variation (6.29%) was not higher than 35%; the coefficient of variation (3.89%) of the mean specific growth rate did not exceed 7%

Conclusions:

After 72 hours of exposure no effects have been observed up to the limit of the water solubility. The respective 72-hour ErL50 was above the highest test concentration of 0.44 mg test item/L based on geometric mean measured concentrations, which is equivalent to a nominal loading of 100 mg test item/L. The NOELR for growth rate was set to be above or equal to 0.44 mg test item/L, corresponding to a nominal loading of 100 mg test item/L.

Executive summary:

A study was performed to determine the toxicity of 1,4-Dichloro-5,8-dihydroxyanthraquinone on the growth of the unicellular freshwater green alga Raphidocelis subcapitata. Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201. The nominal test concentrations were prepared with sterile growth medium under sterile conditions. Due to the very low water solubility of the test item (0.0118 mg/L at 20°C) a Water Accommodated Fraction (WAF) of the test item was prepared according to the OECD guidance No. 23. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: Control, 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. The concentrations of the test item in the media were confirmed by measurements of the test item concentrations before and after the test by HPLC-MS/MS (LOQ = 2.236 μg/L). At test start the measured concentrations were 0.041, 0.080, 0.157, 0.332 and 0.568 mg test item/L (0.57 – 0.66 % of nominal). After 72-hours, the measured test item concentrations were 0.001, 0.004, 0.044, 0.135, 0.339 mg test item/L (0.02 – 0.34 % of nominal; 2.76 – 59.59 % of initial). The measured concentration at test initiation were not between 80 – 120 % of nominal and at test initiation the test item concentration decreased by more than 20 %. Therefore, the evaluation of the test was based on the geometric mean measured concentrations of 0.007, 0.019, 0.083, 0.212 and 0.439 mg test item/L. No concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations. The respective 72-hour ErL50 was above the highest test concentration of 0.44 mg test item/L based on geometric mean measured concentrations, which is equivalent to a nominal loading of 100 mg test item/L. The NOELR for growth rate was set to be above or equal to 0.44 mg test item/L, corresponding to a nominal loading of 100 mg test item/L. No effects have been observed up to the limit of the water solubility. All validity criteria were fulfilled as required. This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

Description of key information

After 72 hours of exposure no effects have been observed up to the limit of the water solubility. The respective 72-hour ErCL50 was above the highest test concentration of 0.44 mg test item/L based on geometric mean measured concentrations, which is equivalent to a nominal loading of 100 mg test item/L. The NOELR for growth rate was set to be above or equal to 0.44 mg test item/L, corresponding to a nominal loading of 100 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.44 mg/L

EC10 or NOEC for freshwater algae:

0.44 mg/L

Additional information

"Should read: 72 h-EL 50 (growth rate): >0.44 mg/L; 72 h-NOELR (growth rate): >0.44 mg/L (The results are expressed in terms of geometric mean measured values)"