6-amino-2-(2,4-dimethylphenyl)-1H-benz[de]isoquinoline-1,3(2H)-dione

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:

06 January 2017 to 30 March 2017

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- A correction factor of 1.031 was used. Concentrations are expressed as active ingredient.
- Weighing and preparation of test solutions was performed under dimmed light.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control. In addition, the filter used to prepare the saturated solution (SS) was retained for possible analysis of the residue. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the highest concentration but without algae and samples for analysis were taken at the start and at the end of the test period.
- Sampling method: At t = 0 and t = 72 h, 3 mL was sampled. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15 °C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with a loading rate of 100 mg a.i./L applying three days of magnetic stirring to reach the maximum dissolution of the test material in medium. The obtained aqueous mixture was filtered through a 0.45 μm membrane filter (RC55, Whatman) and the resulting saturated solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and increasingly yellow with increasing concentration at the end of the preparation procedure.
After preparation of the test solutions for the final test, volumes of 30 mL were added to each replicate of the respective test concentration. Subsequently, 0.6 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Cultures were maintained for 3 days prior to the test

ACCLIMATION
- Culturing media and conditions (same as test or not): Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 to 24 °C. Light intensity was 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Stock culture medium was M1. 3 days before the start of the test, cells from the algal stock culture were inoculated in pre-culture medium M2 at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22 to 24 °C

pH:

7.3 to 8.0

Nominal and measured concentrations:

- Nominal concentrations: 1.0, 3.2, 10, 32 and 100 % of a SS at 100 mg a.i./L
- Measured concentrations: 0.015, 0.036, 0.10, 0.35 and 1.1 mg a.i./L (average)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL
- Type: Capped
- Material, size, headspace, fill volume: Plastic culture vessels, containing 30 mL of test solution
- Aeration: No, however algal cells were kept in suspension by continuous shaking
- Initial cell density: 1 x 10^4 cells per mL (provided by adding 0.6 mL of algal suspension)
- Control end cells density: 2.68 x 10^6
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 or 2 replicates of each test concentration without algae.
- No. of vessels per control (replicates): 6 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L,CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L and NaHCO3 50 mg/L.
- Ca/mg ratio: Hardness (Ca + Mg) 0.24 mmol/L (24 mg CaCO3/L)
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature was continuously monitored in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 99 to 107 μE.m^-2.s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Other: At the end of the test microscopic observations were performed on 10 % of the SS and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: 0, 1.0, 10 and 100 % of a SS prepared at 100 mg a.i./L
- Results used to determine the conditions for the definitive study: Yes. The expected EC50 for growth rate inhibition was between concentrations obtained in 10 and 100 % of a SS prepared at 100 mg a.i./L. The expected EC50 for yield inhibition was between concentrations obtained in 1.0 and 10 % of the SS. The test material is a coloured substance and may substantially reduce the amount of light reaching exposed algal cells. As the prime objective of aquatic toxicity tests is to determine the inherent toxicity of a substance, test conditions in the final test were adjusted in order to increase light penetration and minimise the indirect effects of light shielding on algal growth.

DATA HANDLING
- Comparison of average growth rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (lnXj - lnXi) / (tj - ti) (day^-1)
where:
μi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = [(µC - µT) / µC] x 100
where:
%Ir = percent inhibition in average specific growth rate
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

-Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = [(YC - YT) / YC] x 100
where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 0.33 to 0.36 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.057 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95 % confidence interval 0.056 to 0.058 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Details on results:

Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 2 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarised in Table 3.
Inhibition of growth rate increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in 69 % inhibition at an average concentration of 1.1 mg a.i./L. Statistically significant inhibition of growth rate was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.10 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.
Inhibition of yield increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in >95 % inhibition at and above an average concentration of 0.35 mg a.i./L. Statistically significant inhibition of yield was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.036 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.10 mg a.i./L (10 % SS) when compared to the control.

Samples taken from 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg a.i./L were analysed. The measured concentrations were 0.022, 0.048, 0.13, 0.41 and 1.3 mg a.i./L at the start of the test. During the exposure period the measured concentration of the four lowest test concentrations decreased and were 46 to 72 % of initial at the end of the test. The measured concentrations of the highest test concentration remained stable and was 81 % of initial at the end of the test. It was not clear why the test concentrations remained stable during the combined limit/range-finding test and were not stable during the final test. Given these results, the NOEC and EC values were calculated using the average concentrations.
The measured concentrations in the samples taken from 10 % of the SS with and without algae were similar, indicating that the presence of algae did not affect the test concentration.

Acceptability of the test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 268).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 10 %).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 0.5 %).

Results with reference substance (positive control):

The reference test was carried out to check the sensitivity of the test system as used by the testing facility. Algae were exposed for 72 hours to K2Cr2O7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4.
Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72 h ERC50) was 1.3 mg/L with a 95 % confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72 h ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72 h EYC50) was 0.45 mg/L with a 95 % confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72 h EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test Procedure, α = 0.05, one-sided, smaller) or significant inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α = 0.05, one-sided, smaller). Additionally, the EC10 and EC20 were determined.
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average concentrations of the test material.
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Percentage inhibition of growth rate (total test period) during the final test

Average concentration (mg a.i./L)	Group Mean Growth Rate	Std. Dev.	n	% Inhibition
Control	1.864	0.0101	6	-
0.015	1.833	0.0054	3	1.7#
0.036	1.785	0.0105	3	4.2#
0.10	1.282	0.0142	3	31.2*
0.35	0.871	0.0078	3	53.2*
1.1	0.579	0.0594	3	68.9*

*Effect statistically significant

#Effect statistically significant but biologically insignificant (<10 %)

 

Table 2: Percentage inhibition of growth rate at different time intervals during the final test

Average concentration (mg a.i./L)	n	0 to 24 h		24 to 48 h		48 to 72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	6	2.073	-	1.753	-	1.766	-
0.015	3	2.011	3.0	1.750	0.1	1.736	1.7
0.036	3	1.972	4.9	1.721	1.8	1.662	5.9
0.10	3	1.981	4.5	1.150	34.4	0.714	59.6
0.35	3	1.695	18.3	0.598	65.9	0.322	81.8
1.1	3	1.198	42.2	0.082	95.3	0.457	74.1

 

Table 3: Percentage inhibition of yield during the final test

Average concentration (mg a.i./L)	Group Mean Yields	Std. Dev.	n	% Inhibition
Control	267.4	8.17	6	-
0.015	243.1	3.98	3	9.1#
0.036	210.7	6.58	3	21.2*
0.10	45.8	1.97	3	82.9*
0.35	12.7	0.32	3	95.3*
1.1	4.7	1.04	3	98.2*

*Effect statistically significant

#Effect statistically significant but biologically insignificant (<10 %)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material inhibited growth rate significantly at and above average exposure concentrations of 0.10 mg a.i./L. The 72 h-EC50 for growth rate inhibition (ERC50) was 0.35 mg a.i./L with a 95 % confidence interval ranging from 0.33 to 0.36 mg a.i./L. The 72 h-NOEC for growth rate inhibition was 0.036 mg a.i./L.

Executive summary:

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3 and the OECD series on testing and assessment number 23 under GLP conditions.

The test material was not completely soluble in test medium at the loading rate initially prepared. Weighing and preparation of test solutions was performed under dimmed light. A saturated solution (SS) was prepared at a loading rate of 100 mg a.i./L and used as a stock solution. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg a.i./L. The initial algal cell density was 10^4 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the startand 72 hours of exposure.

Samples taken from 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg a.i./L were analysed. The measured concentrations were 0.022, 0.048, 0.13, 0.41 and 1.3 mg a.i./L at the start of the test. During the exposure period the measured concentration of the four lowest test concentrations decreased and were 46 to 72 % of initial at the end of the test. The measured concentrations of the highest test concentration remained stable and was 81 % of initial at the end of the test. Given these results, the NOEC and EC values were calculated using the average concentrations, i.e. 0.015, 0.036, 0.10, 0.35 and 1.1 mg a.i./L.

Inhibition of growth rate increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in 69 % inhibition at an average concentration of 1.1 mg a.i./L. Statistically significant inhibition of growth rate was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.10 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.

Inhibition of yield increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in >95 % inhibition at and above an average concentration of 0.35 mg a.i./L. Statistically significant inhibition of yield was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.036 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at and above average exposure concentrations of 0.10 mg a.i./L. The 72 h-EC50 for growth rate inhibition (ERC50) was 0.35 mg a.i./L with a 95 % confidence interval ranging from 0.33 to 0.36 mg a.i./L. The 72 h-NOEC for growth rate inhibition was 0.036 mg a.i./L.

The 72 h-EC50 for yield inhibition (EYC50) was 0.057 mg a.i./L with a 95 % confidence interval ranging from 0.056 to 0.058 mg a.i./L. The 72 h-NOEC for yield inhibition was 0.015 mg/L.

Description of key information

Under the conditions of this study, the test material inhibited growth rate significantly at and above average exposure concentrations of 0.10 mg a.i./L. The 72 h-EC50 for growth rate inhibition (ERC50) was 0.35 mg a.i./L with a 95 % confidence interval ranging from 0.33 to 0.36 mg a.i./L. The 72 h-NOEC for growth rate inhibition was 0.036 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.35 mg/L

Additional information

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3 and the OECD series on testing and assessment number 23 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was not completely soluble in test medium at the loading rate initially prepared. Weighing and preparation of test solutions was performed under dimmed light. A saturated solution (SS) was prepared at a loading rate of 100 mg a.i./L and used as a stock solution. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg a.i./L. The initial algal cell density was 10^4 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the startand 72 hours of exposure.

Samples taken from 1.0, 3.2, 10, 32 and 100 % of the SS prepared at 100 mg a.i./L were analysed. The measured concentrations were 0.022, 0.048, 0.13, 0.41 and 1.3 mg a.i./L at the start of the test. During the exposure period the measured concentration of the four lowest test concentrations decreased and were 46 to 72 % of initial at the end of the test. The measured concentrations of the highest test concentration remained stable and was 81 % of initial at the end of the test. Given these results, the NOEC and EC values were calculated using the average concentrations, i.e. 0.015, 0.036, 0.10, 0.35 and 1.1 mg a.i./L.

Inhibition of growth rate increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in 69 % inhibition at an average concentration of 1.1 mg a.i./L. Statistically significant inhibition of growth rate was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.10 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.

Inhibition of yield increased with increasing concentration of the test material from 0.015 mg a.i./L upwards resulting in >95 % inhibition at and above an average concentration of 0.35 mg a.i./L. Statistically significant inhibition of yield was found at test concentrations of 0.015 mg a.i./L and higher. However, only the inhibition observed at 0.036 mg a.i./L and higher was biologically relevant, i.e. inhibition was ≥ 10 %.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at and above average exposure concentrations of 0.10 mg a.i./L. The 72 h-EC50 for growth rate inhibition (ERC50) was 0.35 mg a.i./L with a 95 % confidence interval ranging from 0.33 to 0.36 mg a.i./L. The 72 h-NOEC for growth rate inhibition was 0.036 mg a.i./L.

The 72 h-EC50 for yield inhibition (EYC50) was 0.057 mg a.i./L with a 95 % confidence interval ranging from 0.056 to 0.058 mg a.i./L. The 72 h-NOEC for yield inhibition was 0.015 mg/L.

A supporting study is also available in which the acute toxicity of the test material was calculated using ECOSAR v1.1 (2012) 2000 U.S. Environmental Protection Agency. Given that the substance is an organic molecule within the Molecular Weight range and Log Kow range of the training set compounds, the prediction is considered to be acceptable. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute toxicity of the test material to green algae was calculated to be 0.086 mg/L.