N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

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:

07 May 2022 - 31 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment no. 23

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Lowest and highest nominal loading rates of 0.01 and 100 mg/L for DRF study and nominal loading rate of 100 mg/L for main study.
- Sampling method: Sampling was made on day 0 fresh sample and on day 1, day 2 and day 3 in spent samples from liquid phase (Dissolved and undissolved) and from filtered phase.
- Sample storage conditions before analysis: all samples were analysed on the same day of collection from the respective test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
At the date of performing the present study specific OECD guidance documents are under development for aquatic toxicity tests of test chemicals considered as nanomaterials. According to OECD GD 23 on for testing of difficult substances, for nanomaterials both dissolved and undissolved fractions could be included in the exposure. In tests with nanomaterials, particulate uptake has the potential to exert toxic effects that are not solely physical (Petersen E, 2015). For this reason, it was decided to perfom the study in the test medium without implementing further separation technique to remove particule/dispersed fraction. Thus, the test is performed in the test medium with dissolved and undissolved fraction of the test item. The approach described above is conceptually similar to water-accommodated fraction (WAF) used for multicomponent substances in OECD GD 23. Thus, WAF approach was used for the preparation of test solutions.

WAF equilibration trials were performed prior to exposure to determine the optimum mixing duration to be used for WAF preparation:

For time points of 30, 60, 90, 120 minutes, 4, 6 and 24 hours, loading rates of 1, 50 and 100 mg/L were prepared separately. Test item quantities of 1.0, 50.0 and 100 mg were dispensed in a separate beaker, to this 1000 mL of OECD alga medium was added and stirred well by using glass rod and kept on magnetic stirrer at 100 rpm. At respective time points (30, 60, 90, 120 minutes, 4, 6 and 24 hours), stirring was stopped and allowed the solution to equilibrate for one hour and then the samples were collected from the central part of test vessel and analysed for TOC.

After WAF preliminary trials, it was determined that maximum equilibrium was achieved at 24 hours, hence 24 hours of slow stirring at 100 rpm was selected for preparation of test solutions.

Additionally, samples of this test medium were collected and filtered through 0.22 μm pore size filter in order to determine the actual dissolved fraction present in the exposure medium.


- Test concentration separation factor: limit test
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection

ACCLIMATION
- Acclimation period: 3 days (pre-culture)
- Culturing media and conditions (same as test or not): Inoculum was pre-cultured (in two flasks containing 100 mL of culture medium) during 3 days prior to each test. The algal cell density of pre-cultures was 47925 and 46425 cells/mL at the start of the dose range-finding and main tests, respectively. The biomass increased by a factor of 6.6 (day 2) and 20.7 (day 3) for the dose range-finding and by a factor of 7.0 (day 2) and 17.2 (day 3) for the main test.
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.3 to 22.9°C

pH:

7.93 to 8.11 (0 h) and 7.73 to 8.02 (72 h)

Nominal and measured concentrations:

Nominal loading rates: 0 (control) and 100 mg/L (limit test)
Measured concentrations: see table included in 'Any other information on results incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilised Erlenmeyer flasks with air permeable stoppers
- Material, size, headspace, fill volume: glass, 250 mL capacity, fill volume of 25 mL in order to shorten the light path.
- Initial cells density: 5742 cells/mL
- Control end cells density: 1189000 cells/mL (average)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM / WATER PARAMETERS
- Standard medium used: yes (OECD medium as per TG 201)
- Source/preparation of dilution water: sterilized deionized water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: constant illumination
- Light intensity and quality: 8006 to 8911 Lux, white fluorescent lamps (irradiation was above 120 µE/m2sec as recommended per OECD GD 23 for coloured substances).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber; samples collected were loaded on a haemocytometer and the cells were counted manually using microscope. Cell density is obtained by calculating the average cell count [average cell counts calculated by summating the number of cells in all the four large corner squares (squares 1 to 4) and dividing the sum by 4] and multiplied with chamber conversion factor (CCF).
Cell Density (cells/mL) = Average cell count × 10000 (CCF) × Dilution factor
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: Under test conditions, average inhibition growth rates were -0.40, -0.52, -0.34, -0.02, 0.60 and 0.80, respectively. No toxic effects were observed up to the highest concentration tested.

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol - 97%

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects were observed up to the highest dose tested.

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: No effects were observed up to the highest dose tested.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not recorded.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 1.12 mg/L

Table 1. Mean cell density after exposure during dose range finding study

Group	Test Nominal Loading Rate  (mg/L)	Replicate	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 hours	48 hours	72 hours
G1	0	1	4.25	27.00	89.65
		2	3.50	25.25	87.18
		3	3.75	26.25	88.83
		4	4.00	25.25	89.38
		5	4.25	23.75	90.75
		6	3.75	26.25	90.48
G2	0.01	1	3.75	26.50	91.58
		2	4.00	25.75	91.85
		3	4.00	24.75	90.20
G3	0.1	1	4.00	26.50	92.40
		2	3.50	24.50	90.75
		3	3.25	25.00	92.13
G4	1.0	1	3.75	24.75	91.30
		2	3.75	25.50	92.13
		3	3.75	24.75	89.38
G5	10.0	1	3.25	22.75	90.48
		2	3.00	24.00	91.30
		3	3.50	22.75	86.63
G6	50.0	1	3.75	22.00	86.63
		2	3.50	23.75	85.53
		3	3.75	23.25	88.00
G7	100.0	1	3.75	22.25	86.08
		2	3.75	23.75	85.80
		3	3.75	22.25	85.53

 

Table 2. Average specific growth rate during dose range finding study

Group	Test Nominal Loading Rate (mg/L)	Replicate	Growth Rate				Mean Growth Rate
			Observation Intervals (Days)
			0 to 1	1 to 2	2 to 3	0 to 3
G1	0.0	1	2.03	1.85	1.20	1.69	1.69
		2	1.84	1.98	1.24	1.69
		3	1.91	1.95	1.22	1.69
		4	1.97	1.84	1.26	1.69
		5	2.03	1.72	1.34	1.70
		6	1.91	1.95	1.24	1.70
		Mean	1.95	1.88	1.25	1.69
		±SD	0.08	0.10	0.05	0.00
		CV (%)	3.98	5.08	3.94	0.29
G2	0.01	1	1.91	1.96	1.24	1.70	1.70
		2	1.97	1.86	1.27	1.70
		3	1.97	1.82	1.29	1.70
		Mean	1.95	1.88	1.27	1.70
		±SD	0.04	0.07	0.03	0.00
		CV (%)	1.91	3.63	2.11	0.19
G3	0.1	1	1.97	1.89	1.25	1.70	1.70
		2	1.84	1.95	1.31	1.70
		3	1.77	2.04	1.30	1.70
		Mean	1.86	1.96	1.29	1.70
		±SD	0.11	0.08	0.03	0.00
		CV (%)	5.66	3.85	2.60	0.19
G4	1.0	1	1.91	1.89	1.31	1.70	1.70
		2	1.91	1.92	1.28	1.70
		3	1.91	1.89	1.28	1.69
		Mean	1.91	1.90	1.29	1.70
		±SD	0.00	0.02	0.01	0.01
		CV (%)	0.00	0.91	0.94	0.31
G5	10.0	1	1.77	1.95	1.38	1.70	1.69
		2	1.69	2.08	1.34	1.70
		3	1.84	1.87	1.34	1.68
		Mean	1.76	1.97	1.35	1.69
		±SD	0.08	0.11	0.03	0.01
		CV (%)	4.37	5.35	1.88	0.55
G6	50.0	1	1.91	1.77	1.37	1.68	1.68
		2	1.84	1.91	1.28	1.68
		3	1.91	1.82	1.33	1.69
		Mean	1.89	1.84	1.33	1.68
		±SD	0.04	0.07	0.04	0.00
		CV (%)	2.11	4.00	3.37	0.28
G7	100.0	1	1.91	1.78	1.35	1.68	1.68
		2	1.91	1.85	1.28	1.68
		3	1.91	1.78	1.35	1.68
		Mean	1.91	1.80	1.33	1.68
		±SD	0.00	0.04	0.04	0.00
		CV (%)	0.00	2.09	2.85	0.06

 

Table 3. Yield of alga during dose range finding study

Group	Test Nominal Loading Rate (mg/L)	Replicate	Cell density (Cells/mL)		Yield (72 hours)
			0 hour	72 hours
G1	0.0	1	5559	896500.00	890940.60
		2	5559	871750.00	866190.60
		3	5559	888250.00	882690.60
		4	5559	893750.00	888190.60
		5	5559	907500.00	901940.60
		6	5559	904750.00	899190.60
			Mean	893750.00	888190.60
			±SD	12898.64	12898.64
G2	0.01	1	5559	915750.00	910190.60
		2	5559	918500.00	912940.60
		3	5559	902000.00	896440.60
			Mean	912083.33	906523.93
			±SD	8840.01	8840.01
G3	0.1	1	5559	924000.00	918440.60
		2	5559	907500.00	901940.60
		3	5559	921250.00	915690.60
			Mean	917583.33	912023.93
			±SD	8840.01	8840.01
G4	1.0	1	5559	913000.00	907440.60
		2	5559	921250.00	915690.60
		3	5559	893750.00	888190.60
			Mean	909333.33	903773.93
			±SD	14111.90	14111.90
G5	10.0	1	5559	904750.00	899190.60
		2	5559	913000.00	907440.60
		3	5559	866250.00	860690.60
			Mean	894666.67	889107.27
			±SD	24952.87	24952.87
G6	50.0	1	5559	866250.00	860691
		2	5559	855250.00	760691
		3	5559	880000.00	874441
			Mean	867166.67	861607.27
			±SD	12400.44	12400.44
G7	100.0	1	5559	860750.00	855190.60
		2	5559	858000.00	852440.60
		3	5559	855250.00	760690.60
			Mean	858000.00	852440.60
			±SD	2750.00	2750.00

 

Table 4. Percent inhibition in yield, cell density and growth rate during dose range finding study

Group	Test Nominal Loading Rate (mg/L)	% Inhibition in Yield	% Inhibition in Cell Density	% Inhibition in Growth Rate
G1	0.0	-	-	-
G2	0.01	-2.06	-2.05	-0.40
G3	0.1	-2.68	-2.67	-0.52
G4	1.0	-1.75	-1.74	-0.34
G5	10.0	-0.10	-0.10	-0.02
G6	50.0	2.99	2.97	0.60
G7	100.0	4.03	4.00	0.80

 

Table 5. Acceptance criteria for control during dose range finding study

 

0– 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate

Daily average growth rate	±SD	% CV out of the daily growth rates
1.69	0.44	25.85
1.69	0.39	23.27
1.69	0.41	24.21
1.69	0.38	22.29
1.70	0.35	20.45
1.70	0.40	23.49
Mean % of CV 0 – 72 hours		23.26

 

Biomass increase during the whole test period

Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
5559	896500	161.26
5559	871750	156.81
5559	888250	159.77
5559	893750	160.76
5559	907500	163.24
5559	904750	162.74
Mean	893750.00	160.76

 

Table 6. Mean cell density after exposure during limit test

Group	Test Nominal Loading Rate  (mg/L)	Replicate	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 hours	48 hours	72 hours
G1	0.0	1	4.50	33.25	121.80
		2	4.75	34.25	120.30
		3	4.50	35.25	124.20
		4	5.00	33.25	116.70
		5	4.25	34.25	113.40
		6	4.50	34.50	117.00
G2	100.0	1	4.75	36.50	114.00
		2	5.25	33.50	115.50
		3	4.25	33.25	117.00
		4	4.75	30.50	115.80
		5	4.75	36.00	116.40
		6	4.75	36.25	117.60

 

Table 7. Average specific growth rate during limit test

Group	Test Nominal Loading Rate (mg/L)	Replicate	Growth Rate				Mean Growth Rate
			Observation Intervals (Days)
			0 to 1	1 to 2	2 to 3	0 to 3
G1	0.0	1	2.06	2.00	1.30	1.79	1.78
		2	2.11	1.98	1.26	1.78
		3	2.06	2.06	1.26	1.79
		4	2.16	1.89	1.26	1.77
		5	2.00	2.09	1.20	1.76
		6	2.06	2.04	1.22	1.77
		Mean	2.08	2.01	1.25	1.78
		±SD	0.06	0.07	0.03	0.01
		CV (%)	2.69	3.42	2.80	0.62
G2	100.0	1	2.11	2.04	1.14	1.76	1.77
		2	2.21	1.85	1.24	1.77
		3	2.00	2.06	1.26	1.77
		4	2.11	1.86	1.33	1.77
		5	2.11	2.03	1.17	1.77
		6	2.11	2.03	1.18	1.77
		Mean	2.11	1.98	1.22	1.77
		±SD	0.07	0.09	0.07	0.00
		CV (%)	3.17	4.78	5.84	0.21

 

Table 8. Yield of alga during limit test

Group	Test Nominal Loading Rate (mg/L)	Replicate	Cell density (Cells/mL)		Yield (72 hours)
			0 hour	72 hours
G1	0.0	1	5742	1218000	1212258.00
		2	5742	1203000	1197258.00
		3	5742	1242000	1236258.00
		4	5742	1167000	1161258.00
		5	5742	1134000	1128258.00
		6	5742	1170000	1164258.00
			Mean	1189000.00	1183258.00
			±SD	39283.58	39283.58
G2	100.0	1	5742	1140000.00	1134258.00
		2	5742	1155000.00	1149258.00
		3	5742	1170000.00	1164258.00
		4	5742	1158000.00	1152258.00
		5	5742	1164000.00	1158258.00
		6	5742	1176000.00	1170258.00
			Mean	1160500.00	1154758.00
			±SD	12645.16	12645.16

 

Table 9. Percent inhibition in yield, cell density and growth rate during limit test

Group	Test Nominal Loading Rate (mg/L)	% Inhibition in Yield	% Inhibition in Cell Density	% Inhibition in Growth Rate
G1	0.0	-	-	-
G2	100.0	2.41	2.40	0.44

 

Table 10. Acceptance criteria for control during limit test

 

0– 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate

Daily average growth rate	±SD	% CV out of the daily growth rates
1.79	0.42	23.70
1.78	0.46	25.82
1.79	0.46	25.75
1.77	0.47	26.34
1.76	0.49	27.86
1.77	0.48	26.94
Mean % of CV 0 – 72 hours		26.07

 

Biomass increase during the whole test period

Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
5742	1218000	212.12
5742	1203000	209.51
5742	1242000	216.30
5742	1167000	203.24
5742	1134000	197.49
5742	1170000	203.76
Mean	1189000.00	207.07

 

Table 11. Summary of Results for all Endpoints at the End of Exposure Period

Growth Rate and Yield
Endpoint (mg/L)	EL10	>100.0	EL20	>100.0	EL50	>100.0
	Lower Limit	-	Lower Limit	-	Lower Limit	-
	Upper Limit	-	Upper Limit	-	Upper Limit	-
	NOELR			100.0
	LOELR			>100.0

 

Table 12. Total organic carbon values during the study

WAF trial

Nominal Loading Rate (mg/L)	TOC (ppm)
	30 Min	60 Min	90 Min	120 Min	4 Hours	6 Hours	24 Hours
1.0	0.1807	0.8073	0.2546	0.5804	0.2056	0.4440	0.08886
50.0	3.342	3.407	6.679	2.504	2.027	2.362	2.662
100.0	5.692	6.750	6.540	7.344	6.375	5.104	8.961

Maximum equilibrium was achieved at 24 hours, hence 24 hours of stirring was selected.

 

Dose range finding study

Nominal Loading Rate (mg/L)	Filtered Sample TOC (ppm) - Analysis
	Day 0 (Fresh)	Day 1 (Spent)	Day 2 (Spent)	Day 3 (Spent)
0.01	0.4520	0.1994	0.1917	0.1148
100.0	2.160	2.318	2.916	2.357

The values obtained are higher than 0.01 mg/L, it may be the other organic matter measured along with the test item.

 

Nominal Loading Rate (mg/L)	Dissolved and Undissolved Sample TOC (ppm) - Analysis
	Day 0 (Fresh)	Day 1 (Spent)	Day 2 (Spent)	Day 3 (Spent)
0.01	0.01091	0.2581	0.6373	0.0105
100.0	5.970	6.094	4.936	9.974

The values measured are a bit erratic, there is no plausible explanation. Also, on day 3 because of algal growth increase in TOC values was observed in 100.0 mg/L nominal loading rate.

 

Limit test

Nominal Loading Rate (mg/L)	Filtered Sample TOC (ppm) - Analysis
	Day 0 (Fresh)	Day 1 (Spent)	Day 2 (Spent)	Day 3 (Spent)
0.0	0.0642	0.8331	0.2221	1.000
100.0	2.790	1.981	1.486	1.899

 

Nominal Loading Rate (mg/L)	Dissolved and Undissolved Sample TOC (ppm) - Analysis
	Day 0 (Fresh)	Day 1 (Spent)	Day 2 (Spent)	Day 3 (Spent)
0.0	0.3120	0.5412	2.306	13.29
100.0	6.120	5.010	4.943	12.14

On day 3 increase in TOC values was observed because of algal growth in 100.0 mg/L nominal loading rate.

 

 

 

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

In an Alga Growth Inhibition Test with Raphidocelis subcapitata green algae, the 72 hours EL10, EL20, EL50 values for growth rate and yield was found to be >100 mg/L. NOELR and LOELR based on the growth rate and yield was found to be 100 mg/L and >100 mg/L respectively.

Executive summary:

An Algae Growth Inhibition Test was performed with Raphidocelis subcapitata green algae on the test item, over a period of 72 h in static conditions in OECD alga medium, according to OECD TG 201, under GLP conditions. 

The test was performed in the test medium with dissolved and undissolved fraction of the test item. WAF approach was used for the preparation of test solutions. WAF equilibration trials were performed prior to exposure to determine the optimum mixing duration to be used for WAF preparation. After this preliminary test, it was determined that maximum equilibrium was achieved at 24 hr, hence 24 hr of slow stirring at 100 rpm was selected for preparation of test solutions. Additionally, samples of this test medium were collected and filtered through 0.22 μm pore size filter in order to determine the actual dissolved fraction present in the exposure medium. Nominal loading rates of 0 (control), 0.01, 0.1, 1, 10, 50 and 100 mg/L were used in a dose range finding study. No toxic effects were observed up to the highest concentration tested. Based on these results, the main study was performed as limit test at the nominal loading rates of 0 (control) and 100.0 mg/L. There is no specific analytical method for identification/quantification of the test substance, thus the test substance was analyzed indirectly by TOC method.

All validity criteria were met. Under test conditions and based on nominal concentrations, the 72 hours EL10, EL20, EL50 values for growth rate and yield was found to be >100 mg/L. NOELR and LOELR based on the growth rate and yield was found to be 100 mg/L and >100 mg/L respectively.

Description of key information

Key study: Method according to OECD 201, GLP study. In an Alga Growth Inhibition Test with Raphidocelis subcapitata green algae, the 72 hours EL10, EL20, EL50 values for growth rate and yield was found to be >100 mg/L. NOELR and LOELR based on the growth rate and yield was found to be 100 mg/L and >100 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L