4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

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:

From December 13, 2000 to April 27, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guideline and compliant to GLP, well documented translation of original report (in Japanese)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Principles of method if other than guideline:

Deviating from the guideline, growth rate related inhibition of growth was determined for the interval of 24 to 72 hours only, instead of zero to 72 hours, as recommended by the guideline. From evaluating the raw data, this deviation leads to a slight overestimation of growth rate related inhibition and therefore does not put into question the reliability of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

The study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

EC Number:

229-245-3

EC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

Cas Number:

6448-95-9

Molecular formula:

C24H18N4O4

IUPAC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)diazenyl]-N-phenyl-2-naphthamide

Test material form:

not specified

Details on test material:

Name of test material (as cited in study report): Acute Toxicity Test of ... to Japanese killifish (Oryzias la tipes): I. Pigment Red 22 (abbreviated name: PR)
Analytical purity: Over 99%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the initiation of exposure, the samplings were made from the remaining test solutions for all groups after distributing to test vessels. At the termination of exposure, the sampling of about the same volume of test solutions was made from three test vessels of each test group for analysis. After sampling, algae were removed by centrifugation (2500 rpm, 10 minutes), the supernatants were analyzed by HPLC.

Test solutions

Vehicle:

yes

Details on test solutions:

After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was made up to the volume with culturing medium to prepare the stock solution (100 mg/L). After a necessary aliquot was weighed and sampled from the stock solution for the preparation of each test concentration, the shortfall of co-solvent was added to each solution to adjust the constant concentration of co-solvent (100 mg/L) in the test solution and made up with
medium. In the preparation of test solutions, the amount was considered to cover the necessary solutions for pH measurement and analytical sampling, since the sampling volume for analytical samples at the initiation of the exposure was expected to be more than 5% of test solution volume. The stock solution was used as the test solution of 100 mgIL in the test as it is.
The prepared test solutions showed strong red color due to test substance original in proportion to the test concentration but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless without visible undissolved test substance.

Test organisms

Test organisms (species):

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

Details on test organisms:

- Scientific name: Selenastrum capricornutum
- Supplier: American Type Culture Collection
- Strain No.: ATCC22662 strain
- Date of receipt: August 1, 1996
- Maintenance after received: Aseptically sub-cultured using agar slant medium.
- Pre-cultivation period (definitive test): January 19, 2001 - January 22, 2001. During this period, exponential growth of algae was observed (the
environmental conditions were the same as in the test).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Test temperature:

23 deg C for all test vessels

pH:

7.5 to 7.9 for control and treatment groups during the 72 hours of incubation.

Nominal and measured concentrations:

The following nominal test item concentrations were tested (mg/L):
0.23, 1.0, 3.2, 10, 32, 100;
The measured concentrations of the test substance in the test solution were 94 - 100% and 69 - 96% of the nominal at the initiation of exposure and
termination, respectively. The concentration of test substance in the control and solvent control were less than quantification limit (0.06 mg/L) at the initiation and termination of exposure.
Please see table under section "Any other information on results including tables" for details on analytical results.

Details on test conditions:

- Type of exposure: Static, shaking culture (100 rpm)
- Volume of test solution: 100 mL (OECD medium) / vessel
- Replicates: 3 vessels per test group
- Initial cell concentration: 1 x 10^4 cells/mL
- Lighting: Continuous lighting of 4000 - 5000 lx (within +/- 20%, near the liquid level in flask)
- In both of the pre-cultivation and the study, OECD medium recommended by OECD Guideline was used with sterilization and filtration before use.
- Test vessel: 500 mL glass Erlenmeyer flask (air-permeable silicon stopper)
- Algal culturing apparatus: AGP-50RL (Daikin Plant Co., Ltd.; continuous shaking culture. Able to maintain constant lighting intensity during the culturing)
- Hemocytometer: Burker-Turk
- Particle counter: Coulter Multisizer II (Beckman Coulter, Inc.)
- Electrolyte solution for particle counter: ISOTON II (Beckman Coulter, Inc.)
- Illuminometer: SPI-6A (Topcon Corporation)

- Selection of test concentration:
A range-finding test was conducted at three concentration levels of 1.0 and 10 mg/L, and 100 mg/L (which was the maximum attainable homogeneous dispersion concentration using the co-solvent at 100 mg/L). At the termination (72-hr) of exposure, growth inhibitions (by comparison of areas under the growth curve) were 5.1, ca. 60 and ca. 81% at 1.0, 10 and 100 mg/L treatment level, respectively.
Based on these results, the nominal test concentration in the definitive test was fixed to be performed at six concentration levels of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (common ratio: 3.2). As reference, a control group with dilution water only as well as a solvent control group (with solvent concentration of 100 mg/L identical to the treatment groups) were allocated in the study.

- Test operation procedure:
The number of cells of pre-cultivated algae was counted with a particle counter and an aliquot of pre-cultivated solution was added to each test solution to make 1 x 10^4 cels/mL of cell concentration. After mixing well, 10 mL of the solution, each was transferred into three test vessels per group.
The test was started by placing each of the test vessels in a culturing apparatus maintained at 23 +/- 2 deg C. After 24, 48 and 72 hours of exposure, cell concentrations in all three vessels of all groups were measured. A part of test solution was sampled from each test vessel and the cell concentrations were measured with a hemocytometer.
The pH at the initiation of exposure was measured for the remaining test solutions after distributing into test vessels. At the termination of exposure, pH was measured for one vessel (No. 1) out of three vessels in each test group, since difference in growth rate of each test group were within double. The temperature, lighting intensity and rotation speed in algal culturing apparatus were measured once a day during the exposure period.

Growth curve
The growth curve was drawn by plotting the mean cell concentration of each test group against the time.
Calculation of growth inhibition concentration

Growth inhibition rates were calculated in accordance with the methods described below (area method and growth rate method).
1) Growth inhibition rates (IA) by comparison of areas under the growth curve (Area-based method)
Area under the growth curve was calculated using the following equitation:
A = [(N_1 - N_0)/2]*t_1 + [(N_1 + N_2 - 2*N_0)/2]*(t_2 - t_1) + ... + [(N_n-1 + N_n - 2*N_0)/2]*(t_n - t_n-1)
whith
N_0: Nominal cell concentration at the initiation of exposure (cells/mL)
N_i: Measured cell concentration at t_i (cells/mL)
N_n: Measured cell concentration at to (cells/mL)
t_1: Time of first measurement of cell concentration after initiation of exposure
t_n: Time of nth measurement of cell concentration after initiation of exposure
Calculation of inhibition based on area under the growth curve [%]:
I_A = [(A_c - A_t)/A_c]*100
where A_c: Area under the growth curve for solvent control; A_t: Area under the growth curve for the treatment group.
Calculation of growth rate based inhbition was performed according to formulas given in OECD 201.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 97 mg/L gemetric mean measured concentration

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 2.85 mg/L gemetric mean measured concentration

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Test item concentration analytically verified

Basis for effect:

biomass

Remarks:

determined from area under the growth curve

Remarks on result:

other: 95%CI: 14-20

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corresponding to 0.92 mg/L gemetric mean measured concentration

Basis for effect:

biomass

Remarks:

determined from area under the growth curve

Details on results:

Based on results of the preliminary verification for the difference of means of growth in two-sample (Student's t-test) between control and solvent control test group, the solvent control vales were used for statistical analysis, since the growth in solvent control group was increased compared to control group, significantly.
During 72 hours of incubation, the cell concentrations in the control and the solvent control group increased to 305 and 331 fold on average, respectively.
It was observed that the growth in the 0.32 mg/L test group was almost the same as that in the solvent control group. However, in the other test groups, decreased growth was observed depending upon the test concentration. In the comparison of 24-48 hours growth rate, 40% of inhibition was observed at the highest attainable concentration (100 mg/L) compared to solvent control. In case of growth rate during 24 - 72 hours, statistically significat inhibition between 14 and 34% was observed in the test groups exposed to 10 mg/L and above.
The prepared test solutions showed strong red color in proportion to test item concentration, but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless.

Results with reference substance (positive control):

The 50% algal growth inhibition concentration (EbC50) at 72 hr with the reference substance (potassium dichromate, guaranteed grade, Lot No. ACQ2610, Wako Pure Chemical Industries) was 0.50 mg/L, which was comparable to the historical EbC50 of 0.42 - 0.50 mg/L (n=6) obtained at the testing facility since August, 1996.

Reported statistics and error estimates:

The EC50 was determined by the Logit method (biomass based value only).
The no-observed-effect concentration (NOEC) was determined to be the highest test concentration which had no significant difference (a = 0.05) compared with the solvent control group using statistical analysis (homogeneity of variance (Bartlett), one-way analysis of variance and multiple comparison analysis (Dunnett).

Any other information on results incl. tables

Growth rates and rate related inhibition of growth: Selenastrum capricornutum during 72 hour exposure.

Nominal	Vessel	Growth rate per day
Concentr.	No.				Mean	SD	CV		% Inhibition
mg/L		0 to 24 h	24 to 48 h	48 to 72 h	0 to 72 h			24 to 72 h	24 to 72 h
	1	2.0229	1.8014	1.8260	1.8834	0.1214	6.4448	1.8137
	2	2.1725	1.7111	1.8954	1.9263	0.2322	12.0549	1.8033
Control	3	2.2690	1.6430	1.8124	1.9081	0.3238	16.9703	1.7277
	Average	2.1599	1.7141	1.8453	1.9064	Mean CV	11.8233	1.7797
	SD			SD	0.0215
				CV	1.1295
	1	1.8976	2.0381	1.8671	1.9343	0.0912	4.7150	1.9526
	2	2.0516	1.9300	1.8446	1.9421	0.1040	5.3545	1.8873
Solvent Control	3	1.9459	2.0541	1.7741	1.9247	0.1412	7.3359	1.9141
	Average	1.9671	2.0057	1.8285	1.9338	Mean CV	5.8018	1.9180
	SD			SD	0.0087
				CV	0.4492
	1	2.0229	2.0025	1.7193	1.9149			1.8609
	2	2.2332	1.6748	1.8806	1.9295			1.7777
0.32	3	1.9459	2.0812	1.7851	1.9374			1.9332
	Average	2.0748	1.9135	1.7939	1.9274			1.8572	3.2
	SD
	1	2.2214	1.7163	1.9068	1.9481			1.8115
	2	2.0069	1.9858	1.5776	1.8568			1.7817
1	3	1.8810	2.1263	1.5689	1.8587			1.8476
	Average	2.0464	1.9333	1.6924	1.8907			1.8136	5.4
	SD
	1	2.0373	2.1058	1.4679	1.8704			1.7869
	2	1.9459	1.8585	1.5977	1.8007			1.7281
3.2	3	1.9769	1.8733	1.6837	1.8446			1.7785
	Average	1.9874	1.9567	1.5752	1.8398			1.7645	8.0
	SD
	1	2.1199	1.5256	1.3051	1.6502			1.4153
	2	1.7156	1.9454	1.4487	1.7032			1.6971
10	3	1.8810	1.6857	1.4543	1.6737			1.5700
	Average	1.9194	1.7059	1.4040	1.6764			1.5608	18.6**
	SD
	1	1.4907	2.0119	1.2718	1.5915			1.6419
	2	1.4907	1.8903	1.4117	1.5976			1.6510
32	3	1.4398	1.9171	1.3661	1.5743			1.6416
	Average	1.4740	1.9415	1.3482	1.5879			1.6448	14.2**
	SD
	1	1.4398	1.1174	1.4206	1.3259			1.2690
	2	1.4398	1.1328	1.3253	1.2993			1.2290
100	3	1.2698	1.3905	1.2377	1.2993			1.3141
	Average	1.3863	1.2114	1.3275	1.3084			1.2707	33.7**
	SD							1.8137

% Inhibition is related to solvent control, as growth was increased compared to control.

**: Significant difference from solvent control (Dunnett´s Test, alpha = 0.01)

Solvent control: HCO-40 (100 mg/L)

Nominal and measured test item concentrations:

Nominal		Measured Concentration (mg/L)
Concentration (mg/L)	0 Hour	Percent of Nominal	72 Hours	Percent of Nominal
Control	<0.06	--	<0.06	--
Solvent Control	<0.06	--	<0.06	--
0.32	0.32	100	0.22	69
1.0	0.99	99	0.86	86
3.2	3.0	94	2.7	84
10	9.8	98	7.2	72
32	30	94	29	91
100	98	98	96	96

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:
EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);
NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).
These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.
Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.
Thus, observed effects are to be regarded as not relevant because they are
1) most probably not due to intrinsic toxicity of the test item and
2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).
Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.
The test item is to be regarded as non-toxic to freshwater green algae.

Executive summary:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:

EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);

NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).

These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.

Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.

Thus, observed effects are to be regarded as not relevant because they are

1) most probably not due to intrinsic toxicity of the test item and

2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).

Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.

The test item is to be regarded as non-toxic to freshwater green algae.

All validity criteria of the guideline are fulfilled: The average biomass increase in the control culture ant the solvent control was 305 and 331 -fold, respectively. The mean coefficient of variation for section-by-section specific growth rates in control cultures were below 35% (11.8 and 5.8 % for control and solvent control, respectively) and the coefficient of variation of average specific growth rate of replicate control cultures was below 7 % (1.13 and 0.45 % for control and solvent control, respectively).

The robust study summary is based on the translation of the original study report performed by the same company involved with performance of the test (Sumika TechnoService Corporation, Japan).