3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

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

Study period:

Septmeber 2017 - March 2018

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)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The range-finder was perfomed under non-GLP conditions.

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

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. 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. Samples were stored at -20°C in a refrigerator until shipment for analysis. For security reasons, a reserve set of stabilized samples was stored deep frozen at Fraunhofer.

Vehicle:

no

Details on test solutions:

Control:The control consisted of dilution water only. Four replicates with five specimens per replicate were conducted.

Introduction of the test item: Due to the very low water solubility of the test item a Water Accommodated Fraction (WAF) of the test item was prepared according to the OECD guidance No. 23.
For this, a stock solution with a loading of 100 mg/L was prepared, which also served as the highest test concentration. Considering the purity of 97.6%, 102.5 mg test item were transferred into a glass flask that has a bottom draining port, resulting in a nominal loading of 100 mg/L. An adequate amount of Cu-reduced dilution water was added to the bottle and 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 WAFs were filtered before use (0.22 µm PES filter (polyethylensulfone, 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:

The test organisms were young specimens of Daphnia magna, 4 - 24 hours old at test start. Origin of the daphnids is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.

Pre-Treatment:
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.
According to the OECD guideline 202, the daphnids were not fed during the test.

Test type:

static

Water media type:

freshwater

Remarks:

Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a limestone column, and aeration. To avoid copper contamination, plastic water pipes are used.

Limit test:

no

Total exposure duration:

48 h

Hardness:

Ca hardness: 0.8 mmol/L (holding water)
Mg hardness: 0.3 °d
Total hardness: 1.1 mmol/L

Test temperature:

21.0 - 20.5

pH:

8.18 - 8.41

Dissolved oxygen:

7.8 - 8.7 mg/L

Salinity:

Alcalinity: 2.0 mmol/L
NPOC: 4.625 mg/L
No3: 10 mg/L
No2: <0.005 mg/L
NH4: 0.01 mg/L
PO4: 0.67 mg/L
Cl: 0.03 mg/L
Cd: <0.021 (LOD) - <0.064 (LOQ) µg/L
Cr: <0.150 (LOD) µg/L
Cu: <0.87 (LOQ) -0.871 µg/L
Fe: <0.354 (LOD) µg/L
Mn: 0.119 - 0.139 µg/L
Ni: 0.121 (LÖD) - <0.364 (LOQ) µg/L
Pb: <0.796 (LOD) µg/L
Zn: 3.06 - 3.341 µg/L

Conductivity:

264 µS/cm

Nominal and measured concentrations:

Range-finder: 1.0, 10.0 and 100.0 mg test item / L (nominal).
Definite test: control, 6.25, 12.5, 25.0, 50.0 and 100 mg/L (nominal).

Details on test conditions:

Daphnia magna, 4 - 24 h old were exposed to five concentrations of the test item under static conditions for a period of 48 hours. After addition of the test solutions, test specimens were added with a bore Pasteur pipette. The daphnids were exposed without aeration in 50 mL of test solution in numbered glass beakers.
The beakers were covered with glass panes and subjected to a light/dark cycle of 16/8 hours. The test temperature during the test was 20.8°C. Temperature did not vary by more than 1°C within these limits.

Reference substance (positive control):

yes

Remarks:

In order to confirm the sensitivity of the test species Daphnia magna, acute immobilization tests over 24 h with the reference substance (RS) K2Cr2O7 are performed in regular intervals, as proposed by OECD 202.

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

specified: immobilization

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

specified: immobilization

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

specified: immobilization

Remarks on result:

other:

Remarks:

calculated value

Details on results:

At test start and at test termination in all treatments the measured concentrations were below the LOQ. Therefore, the evaluation of the test was based on the nominal loading of the test item.
Neither significant signs of disease nor stress like discolouration or abnormal behaviour were observed in any replicate up to and including the highest test item loading of 100 mg test item/L. All surviving specimens, up to and including the highest test item loading gave the impression of healthy condition.

Results with reference substance (positive control):

In order to confirm the sensitivity of the test species Daphnia magna, acute immobilization tests over 24 h with the reference substance (RS) K2Cr2O7 are performed in regular intervals, as proposed by OECD 202. The results of the latest reference study (November 2017) are in agreement with historical 24 h EC50-values obtained in this institute, and the stated range given in the OECD guideline (EC50 = 0.6 – 2.1 mg/L).
Immobilization after 24 h:
Control: 0 %
0.40 mg RS/L: 0 % related to control: 0 %
0.60 mg RS/L: 0 % related to control: 0 %
0.90 mg RS/L: 5 % related to control: 5 %
1.35 mg RS/L: 50 % related to control: 50 %
2.00 mg RS/L: 75 % related to control: 75 %

24 h EC50 value:
Immobilization: 1.435 mg/L (95% CL :1.23 – 1.75)

Reported statistics and error estimates:

Statistical evaluation: All statistical calculations were based on nominal loadings of the test item. All results and explanations are directly imported from ToxRat.
Lethal concentration for immobility: Effective concentrations were calculated using linear regression (Probit analysis), provided by the computer programme ToxRat Professional®.
NOEC determination for immobility:To justify the use of the Step-down Cochran-Armitage test at first a trend analysis by contrasts using proportions was performed.The linear trend is not significant (p > 0.05). The quadratic trend is not significant (p > 0.05). The analysis of contrasts did not reveal a linear trend, thus the selected Step-down Cochran-Armitage test was replaced by the Bonferroni Fisher test.The NOEC appears to be higher than or equal 100 mg/L.

The concentrations of the test item in the water phase were quantified by chemical analysis performed by the principal investigator . 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 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 test item.

Validity criteria fulfilled:

yes

Remarks:

(1) 0 % the percent mortality observed in the controls over the test period is ≤ 10 %. (2) The dissolved oxygen concentration at the end of the aging period (48 h) was > 3 mg/L in control and test vessels.  

Conclusions:

The test item Macrolex Fluoreszenzrot G is not acutely toxic to Daphnia magna under the chosen test conditions. The 48h-NOEL was set to be ≥ 100 mg/L and the 48h-LOEL was determined to be > 100 mg/L. The 48h-EL50 was calculated to be ≥ 100 mg/L.

Executive summary:

The effects of Macrolex Fluoreszenzrot G on immobilization of Daphnia Magna were investigated according to Guidline OECD 202 and EC method C.2 under static conditions. After 48 hours, 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 (Fraunhofer 2018).

Description of key information

The test item Macrolex Fluoreszenzrot G is not acutely toxic to Daphnia magna under the chosen test conditions. The 48h-NOEL was set to be ≥ 100 mg/L and the 48h-LOEL was determined to be > 100 mg/L. The 48h-EL50 was calculated to be ≥ 100 mg/L.