1,4-dichloro-5,8-dihydroxyanthraquinone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 individual Water Accommodated Fractions (WAFs) of the test item were prepared according to the OECD guidance No. 23 [5].

PREPARATION OF THE TEST SOLUTIONS:
- Considering the purity of 77.6 %, 128.9 mg test item were weighed and transferred to 1 liter Cu-reduced dilution water, resulting in a nominal loading of 100 mg/L, which also served as the highest test concentration
- This solution was stirred slowly avoiding bubble and foam formation for about 96 h at room temperature (about 20°C) to achieve maximum solubility of the test item
- To separate insoluble test item from the test medium the WAF was filtered before use by 0.22 µm PES filter (Polyethylene sulfone, Nalgene, bottle top filter)
- This test solution was diluted with Cu-reduced dilution water to obtain the other test concentrations
- The test solutions were freshly prepared before test start

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM:
- Origin: The organisms were originally supplied by German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene
- Age of parental daphnids: at least 3 weeks old
- Age of the daphnids at the beginning of the test:4 - 24 hours

PRE-TREATMENT AND HOLDING CONDITIONS:
- Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving.
- Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week
- During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL)
- Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium
- 30 mL of this suspension was given to 1 L Daphnia medium
- The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded
- Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens
- Holding- and dilution-water: Purified drinking water was used as holding- and dilution water
- Feeding: None during the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

70–120 mg/L as CaCO3

Test temperature:

21 °C

pH:

8.14 - 8.44

Dissolved oxygen:

7.73 - 8.57 mg/L

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

At test start test concentrations could only be measured for the two highest test concentrations which were 0.003 and 0.005 mg/L. At test start in the lower three test concentrations and at test termination, in all treatments the measured concentrations were below the LOQ of 0.003 mg/L.

Details on test conditions:

EXPOSURE CONDITIONS:
- Volume of test solution per test vessel: 50 mL
- Test medium: medium M4 (OECD 2004)
- Adjustment of pH in the medium: none
- Application of test item: test item dissolved in Medium M4 applied once at the beginning of the test
- Aeration of test vessels: none
- Test item in nominal loadings: 5 (6.25, 12.5, 25.0, 50.0 and 100 mg active substance per liter)
- Renewal of the test solution during the test period: static, no renewal
- Feeding during exposure: none
- Temperature (target): 18 to 22°C, constant within a range of 2°C
- Light intensity (target): 814–816 lx (corresponding to 10.9 μE/(m² * s))
- Light regime: 16 h light; 8 h dark
- Biological parameter: immobility
- Observation and biological measurements: visually determined daily and dead specimens were removed

WATER QUALITY MEASUREMENTS:
At start of the test and at the end of the test the following parameters were measured:
- Temperature
- pH
- Dissolved oxygen content in mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The concentration of the test item in the test solutions were analyzed in each test vessel at commencement of the test and at the end of the test period using HPLC-MS/MS. At test start test concentration could only be measured in the two highest test concentrations with a nominal loading of 50.0 and 100 mg/L. However, at test start in the three lowest test concentrations of 6.25, 12.5 and 2.0 mg/L and at test termination in all treatments the measured concentrations were below the LOQ of 0.003 mg/L (Table 1). Therefore, the evaluation of the test was based on the nominal loading of the active substance.

Results with reference substance (positive control):

The sensitivity of the test clone was checked twice a year in non-GLP tests by using K2Cr2O7 as reference substance. The latest 24h-EC50 (November 2017) was 1.435 mg/L (95 % confidence limits: 1.233–1.745 mg/L).

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore, the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Reported statistics and error estimates:

Since no inhibition of mobility of daphnids was observed during the test, the EL50 was reported as > highest nominal test concentration. The NOEL and LOEL were reported as ≥ highest concentration tested and > highest nominal concentration tested, respectively. All statistical calculations were performed using the computer programme ToxRat® [8].

Validity criteria fulfilled:

yes

Remarks:

immobilisation and other abnormalities in the controls did not exceed 10% by the end of the test; dissolved oxygen concentration remained above 3 mg/L throughout the exposure period

Conclusions:

No significantly increased immobility of daphnids was observed up to and including the highest nominal loading of 100 mg/L when compared to the control daphnids. Consequently, the NOEL was set to be ≥ 100 mg/L and the LOEL was estimated to be > 100 mg/L. The EL50 was determined to be ≥ 100 mg/L. The test was conducted under static conditions for 48 h.

Executive summary:

A study was performed to determine the influence of the test item 1,4-Dichloro-5,8-dihydroxyanthraquinone on immobilization of daphnids of the species Daphnia magna. Considering the test item purity of 77.6%, the daphnids were placed in water containing the test item in nominal loadings of 6.25, 12.5, 25.0, 50.0 and 100 mg active substance per liter. The test was conducted under static conditions for 48 h. Effects on immobilization were determined after 24 and 48 hours according to OECD guideline 202. Samples for chemical analysis were taken of fresh and aged test solutions. The nominal test concentrations were prepared in Cu-reduced dilution water. Due to the very low water solubility of the test item a stock solution was prepared as Water Accommodated Fraction (WAF) of the test item according to the OECD guidance No. 23 and serially diluted to obtain the lower test concentrations. 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 = 0.003 mg/L). At test start test concentrations could only be measured for the two highest test concentrations which were 0.003 and 0.005 mg/L. At test start in the lower three test concentrations and at test termination, in all treatments the measured concentrations were below the LOQ of 0.003 mg/L. Therefore, the evaluation of the test was based on the nominal loadings of the test item (based on active substance concentrations). At the end of the test, no significantly increased immobility of daphnids was observed up to and including the highest nominal loading of 100 mg/L when compared to the control daphnids. Consequently, the NOEL was set to be ≥ 100 mg/L and the LOEL was estimated to be > 100 mg/L. The EL50 was determined to be ≥ 100 mg/L. All validity criteria were fullfilled. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute Daphnia study.

Description of key information

No significantly increased immobility of daphnids was observed up to and including the highest nominal loading of 100 mg/L when compared to the control daphnids. Consequently, the NOEL was set to be ≥ 100 mg/L and the LOEL was estimated to be > 100 mg/L. The EL50 was determined to be ≥ 100 mg/L. The test was conducted under static conditions for 48 h.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

"Should read: EC 50 > 100 mg/L (The result is based on nominal loading)"