2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

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:

30 December 2016 to 10 January 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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

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

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, after 24 hours of exposure and at the end of the test period.
- Sampling method: At t = 0, t = 24 and t = 72 h, 3.0 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/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. The highest test concentration was clear and slightly yellow at the end of the preparation procedure. The lower test concentrations were clear and colourless.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 23 °C

pH:

7.8 to 8.0

Nominal and measured concentrations:

- Nominal concentrations: 1.0, 10 and 100 % of a SS prepared at 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL
- Type: Capped
- Material, size, headspace, fill volume: All glass, filled with 50 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
- Control end cell density: 1.53 x 10^6
- No. of vessels per concentration (replicates): 6 replicates high concentration, 3 replicates lower concentrations; 1 extra replicate for sampling purposes; 1 or 2 replicates without algae.
- No. of vessels per control (replicates): 6 replicates; 1 extra replicate for sampling purposes; 1 or 2 replicates without algae.

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 82 to 86 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 immersion probe (path length =20 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. Cell densities were recorded at 24-hour intervals in the control and the highest test concentration.
- Other: At the end of the test microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: Combined limit/range finding test

DATA HANDLING
- Calibration curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- 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.:

> 100 other: % SS prepared at 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % SS prepared at 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1.
Table 2 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 3 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarised in Table 4.
Statistical significance was checked for the highest test concentration. For both growth rate and yield statistically significant inhibition was found. However, the inhibition observed on growth rate was biologically not 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 the highest test concentration when compared to the control.
Table 5 shows the effect parameters based on loading rate. Measured concentrations in the saturated solution prepared with this loading rate were below 0.01 mg/L.

Samples taken from 100 % of the SS during the range-finding test were analysed but concentrations proved to be below the limit of detection (LOD was 0.01 mg/L) from the start. The low water solubility of the test material and the even lower expected solubility in M2 medium falls below the LOD of the analytical method and therefore precludes the confirmation of test material concentration within the test. However, there were no biologically relevant effects on the growth rate of algal cells observed during the test, thus the test material cannot be considered to have any measurable toxicological effect.

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. 153).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 8 %).
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. 3 %).

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 highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α = 0.05, one-sided, smaller).
No EC50 values could be determined because the observed effects were below 50 %.
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Mean cell densities (x10^4 cells/mL)

Time (h)	% SS at 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	6.2	n.m	n.m	5.0
48	30.7	n.m	n.m	22.8
72	153.4	157.3	149.0	115.0

n.m. = Not measured

 

Table 2: Percentage inhibition of growth rate (total test period)

% SS at 100 mg/L	Mean	Std. Dev.	N	% Inhibition
Control	1.675	0.0458	6	-
1.0	1.686	0.0120	3	-0.6
10	1.666	0.0484	3	0.6
100	1.573	0.0867	6	6.1*

*Effect was statistically significant but biologically not relevant (<10 %)

 

Table 3: Percentage inhibition of growth rate at different time intervals

% SS at 100 mg/L	n	0 to 24 h		24 to 48 h		48 to 72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	6	1.812	-	1.605	-	1.608	-
1.0	3	0.000	100.0	0.000	100.0	5.058	-214.5
10	3	0.000	100.0	0.000	100.0	4.997	-210.7
100	6	1.592	12.2	1.495	6.8	1.631	-1.4

 

Table 4: Percentage inhibition of yield

% SS at 100 mg/L	Mean	Std. Dev.	N	% Inhibition
Control	152.4	19.48	6	-
1.0	156.3	5.70	3	-2.6
10	148.0	21.89	3	2.9
100	114.0	27.58	6	25.2*

*Effect was statistically significant

 

Table 5: Effect parameters

	Parameter, Loading rate (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	100	>100	>100	>100
Yield	Value	<100	<100	<100	>100

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/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. A saturated solution (SS) was prepared at a loading rate of 100 mg/L and used as the test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

A combined limit range-finding test was performed exposing six replicates of exponentially growing algal cultures per group to a control and 100 % of the SS prepared at 100 mg/L under static conditions. In addition, three replicates per group were exposed to 1.0 and 10 % of the SS. 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 start, after 24 and 72 hours of exposure.

Samples taken from 100 % of the SS during the range-finding test were analysed but concentrations proved to be below the limit of detection (LOD was 0.01 mg/L) from the start.

Due to the low solubility of the test material in water, concentration levels that might be toxic for algae could not be reached. The 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

The test material inhibited growth rate and yield by 6 and 25 %, respectively. The 72 h NOEC was at the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

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

Under the conditions of this study, the 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

Description of key information

Under the conditions of this study, the 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.01 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. A saturated solution (SS) was prepared at a loading rate of 100 mg/L and used as the test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

A combined limit range-finding test was performed exposing six replicates of exponentially growing algal cultures per group to a control and 100 % of the SS prepared at 100 mg/L under static conditions. In addition, three replicates per group were exposed to 1.0 and 10 % of the SS. 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 start, after 24 and 72 hours of exposure.

Samples taken from 100 % of the SS during the range-finding test were analysed but concentrations proved to be below the limit of detection (LOD was 0.01 mg/L) from the start.

Due to the low solubility of the test material in water, concentration levels that might be toxic for algae could not be reached. The 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

The test material inhibited growth rate and yield by 6 and 25 %, respectively. The 72 h NOEC was at the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

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

Under the conditions of this study, the 72 h EC50 for the inhibition of both growth rate (ERC50) and yield (EYC50) was above the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L. Measured concentrations in this saturated solution were below 0.01 mg/L.

In a supporting study, 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 2.918 mg/L.