4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzenesulphonic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

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:

other: According to OECD 201 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzene-1-sulfonic acid

EC Number:

201-901-3

Cas Number:

89-36-1

Molecular formula:

C10H10N2O4S

IUPAC Name:

4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzene-1-sulfonic acid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at start and end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 500 mg/L was frehsly prepared with dilution water. Dispersion treatment was ultrasound for 15 min at room temperature.
- Eluate: Dilution water
- Differential loading: 5 - 15.8 - 50 - 158 - 500 mg/L (factor: √10) corresponding to
geometric mean measured concentrations of 0.855 - 1.55 - 2.78 -
155 - 501 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
501 3.04 2.95
155 6.45 6.17
2.78 8.17 7.70
1.55 8.18 8.45
0.855 7.98 8.73
Control 8.17 8.84

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1073776 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes

Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 19 61
10 1 5
1 - 1 - 2

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (21.9-42.2)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05. Normality test failed for yield values. No useful transformation of yield values was possible.

Any other information on results incl. tables

NOEC, LOEC and EC - values of pyrazolic acid 4 (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	2.78
LOEC	155
ErC10	12.2 (9.21 – 20.4)
ErC20	17.3 (13.1 – 26.5)
ErC50	30.6 (21.9 – 42.2)
	Inhibition of Yield
NOEC	2.78
LOEC	155
EyC10	4.67 (2.79 – 17.4)
EyC20	7.53 (4.46 – 19.5)
EyC50	13.9 (7.45 – 26.5)

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
501		1		5074		2823	2229	2680
		2		5074		3737	2717	2897
		3		5074		2897	3069	2155
		Mean		5074		3152	2672	2577
155		1		5074		7460	5275	5828
		2		5074		7415	5820	4578
		3		5074		9473	6484	5779
		Mean		5074		8116	5860	5395
2.78		1		5074		28891	238348	989402
		2		5074		32032	240537	1016208
		3		5074		37259	269028	1012083
		Mean		5074		32727	249304	1005898
1.55		1		5074		25467	207671	937439
		2		5074		22749	208787	1036946
		3		5074		32163	271144	1055039
		Mean		5074		26793	229201	1009808
0.855		1		5074		27009	219619	986065
		2		5074		15779	149919	1028803
		3		5074		35410	256457	1053034
		Mean		5074		26066	208665	1022634
Control		1		5074		36521	249496	1073973
		2		5074		27407	212817	1027196
		3		5074		29645	260918	1208276
		4		5074		25082	197938	945270
		5		5074		32593	236486	1080693
		6		5074		40707	273460	1107248
		Mean		5074		31993	238519	1073776

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

                              

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
501	1		-0.213	100		-2394	100
	2		-0.187	100		-2177	100
	3		-0.285	100		-2919	100
	Mean	(+)	-0.228	100	(+)	-2497	100
155	1		0.046	97.4		754	99.9
	2		-0.034	100		-496	100
	3		0.043	97.6		705	99.9
	Mean	(+)	0.018	98.3	(+)	321	100
2.78	1		1.76	1.48		984328	7.89
	2		1.77	0.98		1011134	5.39
	3		1.77	1.05		1007009	5.77
	Mean	(-)	1.76	1.17	(-)	1000824	6.35
1.55	1		1.74	2.49		932365	12.8
	2		1.77	0.60		1031872	3.45
	3		1.78	0.28		1049965	1.75
	Mean	(-)	1.76	1.12	(-)	1004734	5.99
0.855	1		1.76	1.54		980991	8.21
	2		1.77	0.75		1023729	4.21
	3		1.78	0.31		1047960	1.94
	Mean	(-)	1.77	0.87	(-)	1017560	4.79
Control	1		1.79			1068899
	2		1.77			1022122
	3		1.82			1203202
	4		1.74			940196
	5		1.79			1075619
	6		1.80			1102174
	Mean		1.78			1068702

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.97	1.92	1.46	1.79	0.283	15.9	16.2
	2	1.69	2.05	1.57	1.77	0.249	14.0
	3	1.77	2.18	1.53	1.82	0.325	17.8
	4	1.60	2.07	1.56	1.74	0.281	16.1
	5	1.86	1.98	1.52	1.79	0.240	13.4
	6	2.08	1.91	1.40	1.80	0.355	19.8
				Mean	1.78
				SD ±	0.03
				CV [%]	1.52

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test substance was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 30.6 (21.9 – 42.2) and 13.9 (7.45 – 26.5) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of the test substance.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 10³cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 5.0 - 15.8 - 50.0 - 158 - 500 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test substance were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations at test start were in the range of 97 – 103 % of the nominal values. At the end of the test the test substance gave recoveries of 93 and 98 % at 158 and 500 mg/L and less than the LOQ of 0.300 mg/L at the concentrations of 5 to 50 mg/L of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.

Table 1:    NOEC, LOEC and EC - values of pyrazolic acid 4 (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	2.78
LOEC	155
ErC10	12.2 (9.21 – 20.4)
ErC20	17.3 (13.1 – 26.5)
ErC50	30.6 (21.9 – 42.2)
	Inhibition of Yield
NOEC	2.78
LOEC	155
EyC10	4.67 (2.79 – 17.4)
EyC20	7.53 (4.46 – 19.5)
EyC50	13.9 (7.45 – 26.5)