2,4-dihydro-5-methyl-2-phenyl-4-(phenylazo)-3H-pyrazol-3-one

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

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

2,4-dihydro-5-methyl-2-phenyl-4-(phenylazo)-3H-pyrazol-3-one

EC Number:

224-330-1

Cas Number:

4314-14-1

Molecular formula:

C16H14N4O

IUPAC Name:

2,4-dihydro-5-methyl-2-phenyl-4-(phenylazo)-3H-pyrazol-3-one

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical evaluation of the saturated solution of the test item and the control was carried out via
HPLC-DAD from freshly prepared media after 0 hours. Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. The recovery rates of the test item at the start was below the limit of quantification. Therefore, no analytical evaluation after 72 h was carried out.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
The saturated solution (1.00 mg/L test item was weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h). The dispersion was stirred for 48 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded.
The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded.
After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

Test concentration
The saturated solution was tested in a limit test.

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 every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 22.0 max.: 23.0, mean value: 22.5

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.95 8.42
Control 8.04 8.60

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- 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: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 1025784 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



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, mean value: 70.6



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.546 0.671 ± 0.413
95 % confidence interval 0.496 - 0.606
Yield Inhibition
EyC50 0.281 0.317 ± 0.139
95 % confidence interval 0.220 - 0.347

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Any other information on results incl. tables

Cell Densities

 

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	4847	35321	191966	993914
	2	4847	33825	191937	970199
	3	4847	21169	165296	705717
	4	4847	23449	198787	803274
	5	4847	22583	197806	835216
	6	4847	34104	188348	1043030
	Mean	4847	28409	189023	891892
Control	1	4847	43307	231681	920939
	2	4847	38703	205377	1093492
	3	4847	37448	217788	933398
	4	4847	41479	220704	1158097
	5	4847	41161	209553	961973
	6	4847	39607	201288	1086805
	Mean	4847	40284	214399	1025784

 

 

 

Evaluation after 72 hours

                  Statistically significant differences of growth rates and yield compared to

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

 

 

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.77	1		989067	3
	2		1.77	1		965352	5
	3		1.66	7		700870	31
	4		1.70	5		798427	22
	5		1.72	4		830369	19
	6		1.79	0		1038183	- 2
	Mean	(-)	1.74	3	(-)	887045	13
Control	1		1.75			916092
	2		1.81			1088645
	3		1.75			928551
	4		1.83			1153250
	5		1.76			957126
	6		1.80			1081958
	Mean		1.78			1020937

 

 

 Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	2.19	1.68	1.38	1.75	0.410	23.4	16.9
	2	2.08	1.67	1.67	1.81	0.235	13.0
	3	2.05	1.76	1.46	1.75	0.295	16.8
	4	2.15	1.67	1.66	1.83	0.279	15.3
	5	2.14	1.63	1.52	1.76	0.329	18.7
	6	2.10	1.63	1.69	1.80	0.259	14.3
				Mean	1.78
				SD ±	0.03
				CV [%]	1.81

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours were 1.00 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 1.00 mg/L, respectively.
All effect levels are given based on the nominal concentration of the test item

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) . The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4847 cells/mL. A saturated solution with a nominal concentration of 1 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditionswere determined to be within the acceptable limits.

The concentrations of the test item the test item in the saturated solution and the control were analytically verified via HPLC-DAD at the start of exposure. The recovery rates ofthe test item at the start were below the System Quantification Limit of the analytical method (0.05 mg/L test item).

All effect values of the test item are given based on the nominal concentration.

 

Table1:  NOEC, LOEC, EC - values of the test item (0-72 hours)

                  based on nominal test item concentrations [mg/L]

                               

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
EyC10	< 1.00
EyC20	> 1.00
EyC50	> 1.00