N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-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

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:

28 July, 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from May 31, 2016

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

The cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures. The concentrations of the test item were confirmed by measuring the test item concentrations in the test solutions at initiation and termination of the 72-hour exposure period. pH and temperature were measured daily. The solution pH was measured in one test vessel per test concentration including the control. At the end of the test, the solution pH values were measured directly in test vessels. The test culture temperature was measured in one additional test vessel. The light intensity was measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in µE m-2 s-1 at level of the test media surface.

Vehicle:

no

Details on test solutions:

To prepare a stock solution 100.00 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 vigorously over the weekend at room temperature (about 20°C) to achieve maximum solubility of the test item. Another flask was filled with growth medium only for the control replicates and stirred in the same way.
After 72-hours the test material appeared to be homogeneously dispersed within the test media.
To separate insoluble test item from the test medium, the WAF of each treatment was filtered through a 0.22 µm PES filter. This test solution was serially 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:

TEST ORGANISM
- Common name: green algae
- Strain: Catalog No 61.81 SAG
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Göttingen
- Method of cultivation: stock cultures are maintained acc. to OECD 201 and Bringmann und Kühn (1980). Prior to testing a pre-culture will be established in standard OECD growth medium to obtain exponentially growing algae for the test. 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:

not reported

Test temperature:

21-24 °C, controlled at ± 2°C
22 °C measured daily in additional vessel

pH:

Controls: 7.59 (test start) - 7.92 (meanvalue of n = 8 controls at test end)
Treatments: 7.57 - 7.60 (test start) to 7.83 - 7.92 (test end)

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: 0, 6.25, 12.5, 25.0, 50.0 and 100.0 mg test item/L
Initial (measured): 0, 2.33, 3.92, 9.48, 18.88 and 40.86 µg test item/L
Initial (measured, purity of 98.3 % considered): 0, 2.37, 3.99, 9.64, 19.20 and 41.56 µg test item/L
final (measured): 0, 2.29, 4.70, 9.14, 19.31 and 38.98 µg test item/L
final (measured, purity of 98.3 % considered): 0, 2.33, 4.79, 9.30, 19.64 and 39.65 µg test item/L
arithmetic mean (measured): 0, 2.35, 4.39, 9.47, 19.42 and 40.61 µg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical glass flasks, sterilized
- Type: covered with air-permeable silicone-sponge caps, sterilized
- Fill volume: 250 mL
- Aeration: no
- Type of flow-through: static test system
- Initial cells density: 10000 cells/mL
- no of replicates: 8 per control and 5 per treatment

GROWTH MEDIUM
- Standard medium used: yes, OECD medium, according OECD 201

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Purified water (Elga PURELAB® Ultra)
- Culture and test medium:same

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: permanent
- Light intensity and quality: 92.33 to 93.77 µE m-2 s-1

TEST CONCENTRATIONS
- Spacing factor: 2
- Range finding study yes
- Test concentrations: 1, 10 and 100 mg test item/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 40.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Lumilux ® Yellow CD397

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 40.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Lumilux ® Yellow CD397

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 40.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Lumilux ® Yellow CD397

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 40.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Lumilux ® Yellow CD397

Basis for effect:

biomass

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. Further details are given in the tables below "any other information on results including tables".

Reported statistics and error estimates:

Values rounded to a smaller degree of precision (number of digits) than used in the actual calculation are presented, differences are therefore possible but within the limits of the experimental accuracy.

Measured concentrations were not within a range of 80 – 120 % of the nominal, but remained stable throughout the 72 hours. The evaluation was based on the arithmetic mean measured concentrations of the test item. The mean values of the cell counts for each concentration plot of the exposure test were used for plotting growth curves. For the growth test, mean average growth rates were calculated (entire exposure period of 0 – 72 h).
For the whole exposure period, calculation of the percent inhibition compared to controls were performed according to the guideline using the growth rate or sectional growth rate [r] and the yield [y]. For the growth test, the percent inhibition values of growth rate and yield were plotted as a function of the test item concentration. Since the test results showed no inhibition above 50 %, they were not statistically analysed to determine an EC50 and EC10 values together with 95 % confidence intervals. The NOEC values were determined using appropriate statistical methods (Dunnett`s Multiple t-test Procedure, significance level 0.05, one-sided smaller). The software ToxRat® was used for statistical evaluations.

Table: Growth rate (G) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Mean measured concentration [µg test item/L]	0-24 h		0-48 h		0-72 h
	G	%I	G	%I	G	%I
Control	1.893	0	1.72	0	1.689	0
2.35	1.904	-0.58	1.717	0.19	1.651	2.30
4.39	1.893	0.04	1.716	0.22	1.674	0.91
9.47	1.896	-0.11	1.659	3.54	1.649	2.36
19.42	1.862	1.64	1.713	0.41	1.666	1.40
40.61	1.858	1.89	1.714	0.33	1.695	-0.32

 

 

Table: Yield (Y) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Mean measured concentration [µg test item/L]	0-24 h			0-48 h		0-72 h
	Y	%I		Y	%I	Y	%I
Control	5.7		0.00	30.3	0.00	158.7	0.00
2.35	5.7		-1.32	30.5	-0.73	141.2	11.05
4.39	5.7		-0.17	30.3	0.03	151.5	4.58
9.47	5.7		-0.21	26.6	12.00	140.1	11.71
19.42	5.4		3.67	29.8	1.52	147.4	7.13
40.61	5.4		3.74	30.6	-0.93	162.6	-2.46

 

 

Validity criteria fulfilled:

yes

Conclusions:

The potential growth(r) and yield(y)-inhibiting effect of the test item Lumilux® Yellow CD397 (N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide) on the unicellular freshwater green alga Raphidocelis subcapitata (former name Pseudokirchneriella subcapitata) was investigated in a static exposure system for 72 h according to OECD 201 (2011). Nominal concentrations of 0, 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L have been prepared in sterile OECD medium and were chemically analysed using LC-MS (LOQ = 0.05 µg a.s./L) at the start and the end of the test. Based on the water accommodated fraction (WAF) of the test item and its arithmetic mean measured concentrations of 0, 2.35, 4.39, 9.47, 19.42 and 40.61 µg test item/L, the 72h-ErC50 and EyC50 were determined to be > 40.6 µg test item/L (corresponding to the maximum solubility of the test item in test solution). The NOEC (growth rate and yield) was determined to be ≥ 40.6 µg test item/L. All validity criteria of the test were met.

Description of key information

In a GLP- and guideline-conform (OECD 201, 2011) growth(r) and yield(y)-inhibition test with green algae Raphidocelis subcapitata (former name Pseudokirchneriella subcapitata) and test item Lumilux® Yellow CD397, the 72h-ErC50 and EyC50 were determined to be > 40.6 µg test item/L (WAF, arithmetic mean) and the NOEC (growth rate and yield) was determined to be ≥ 40.6 µg test item/L (WAF, arithmetic mean) (Schlich, 2018). All concentrations correspond to the maximum solubility of the test item in test item solution. All validity criteria of the test were met and hence, the obtained results are considered reliable.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L