4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

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:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although th study was conducted according to EU Method C.3 and in accordance with the Principles of Good Laboratory Practices (GLP), poor solubility issues with the test material did not permit stability of the tested concentrations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test substance concentrations, duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations, the undiluted filtrate and all test dilutions and from the control.
For the determination of the stability of the test substance under the test conditions, sufficient volumes of all freshly prepared test media and the control were incubated under the same conditions as in the test itself (but without algae) and sampled in duplicate at the end of the test (after 72 hours test period).
The concentrations of the test substance MORTRACE SB CONCENTRATE were analysed in all duplicate test media samples from both sampling dates (0 and 72 hours) immediately after sampling. From the control samples only one of the duplicate samples were analysed from each of both sampling dates (0 and 72 hours).

Vehicle:

yes

Details on test solutions:

Just before use of the test substance it was homogenised by warming up in the test substance container to 60°C for 2 hours. Then the test substance was homogenised by intense mixing.
Due to the very low water solubility of the test substance the following dosage of the test substance was chosen:
A supersaturated stock suspension of the test substance with a nominal concentration of 100 mg/I was prepared by weighing 150 mg of the test substance into 1500 ml test water. No auxiliary solvent or emulsifier was used. This supersaturated stock suspension was stirred by a magnetic stirrer at room temperature in the dark over 72 hours to dissolve respectively disperse a maximum concentration of the test substance in the supersaturated stock suspension. The stirring period of 72 hours was chosen according to the results of a pre-test (without GLP), where the concentration of the test substance had slightly increased from 24 hours of stirring to 72 hours of stirring.
The supersaturated stock suspension of the test substance was filtered through a folded filter paper (Schleicher & Schuell, Type 1573 ) after the 72 hours stirring period just before the start of the test. The first 100 ml filtrate were discarded to avoid a loss of the test substance due to adsorption onto the filter. The undiluted filtrate of the supersaturated stock suspension was used as the highest concentrated test medium. Additionally, adequate volumes of the filtrate were diluted with test water for the preparation of the test media with lower test substance concentrations. No additional dilution step was inserted. In this way the following test concentrations were prepared: dilutions 1:3.2, 1:10, 1:32 and 1: 100 of the filtrate (1 part of the filtrate added to 2.2, respectively 9, 31, and 99 parts of test water). The enlarged spacing factor of 3.2 between the test dilutions was chosen, since according to the results of the range-finding test a large concentration range had to be tested. The real concentration of the test substance in the test dilutions were analytically determined.
Additionally, a control was tested in parallel (test water without addition of the test substance).
The test concentrations were based on the results of a range-finding test. However, concentrations far above the water solubility limit of the test substance have not been be tested according to the Commission Directive 92/69/EEC. The range-finding test was not performed in compliance with GLP-Regulations, but the raw data of the range-finding test will be archived under the RCC Project number of the present study.
Then, at the start of the test the algae were inoculated by adding an adequate volume from the pre-culture to the test media.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicalus CHODAT, Strain No.86.81 SAG, supplied by the “Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Gottingen”, D-37073 Gottingen. The algae were grown in the laboratories of RCC under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

Calculated water hardness of the test water: 0.24 mmol/l (≈ 24 mg/I) as CaCO3

Test temperature:

23.2 - 23.4°C

pH:

7.9 - 10.3

Dissolved oxygen:

no information available

Salinity:

not applicable

Nominal and measured concentrations:

The analytically determined test substance concentrations in the samples from the freshly prepared test medium of the highest test concentration (the undiluted filtrate of the supersaturated stock suspension) amounted to 1.9 µg/I. At the lower concentrated test media lower test substance concentrations were measured according to the dilution steps down to 0.025 µg/I at the dilution 1:100
In all test media, incubated under the test conditions (but without algae), the concentrations of MORTRACE SB CONCENTRATE strongly decreased during the test period to values below the determination limit of the analytical method of 0.02 µg/l. Thus, the test substance was obviously not stable under the test conditions, possibly due to the high light intensity, which is necessary in this algal growth inhibition test, Therefore, all biological results are related to the mean measured test substance concentrations (calculated as the average over all measurements during the test period)

Details on test conditions:

Experimental conditions -
The algae were cultivated and tested in synthetic test water, prepared according to the mentioned test guidelines: in deionized water with a conductivity lower than 0.1 µS cm-1 (MilliQ-water) analytical grade salts were added to following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/I
CaCI2 x2H20 18.0mg/I
NH4CI 15.0 mg/I
MgSO4 x 7 H20 15.0 mg/I
MgCl2 x 6H20 12.0 mg/I
KH2PO4 1.6 mg/I
Trace elements:
Na2EDTA x 2 H20 100.0 µg/I
FeCI3 x 6H20 80.0 µg/I
MnC12 x 4 H20 415.0 µg/I
H3B03 185.0 µg/I
Na2MoO4 x 2 H20 7.0 µg/I
ZnCI2 3.0 µg/I
CoCI2 x 6 H20 1.5 µg/I
CuCl2 x 2 H20 0.01 µg/I
Calculated water hardness of the test water: 0.24 mmol/l (= 24 mg/I) as CaCO3
The test was started (0 hours) by inoculation of a biomass of 10000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up about 72 hours prior to the test at the same conditions as in the test.
The test design included three replicates per test concentration and six replicates in the control. Per replicate 50 ml algal suspension were continuously stirred by magnetic stirrers in 50 ml Erlenmeyer flasks. The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath, and continuously illuminated at the measured light intensity of about 8500 Lux (mean value), range: 8000 to 8800 Lux (minimum and maximum value of measurements at 3 places distributed over the experimental area at the level of the test media). This illumination was achieved by fluorescent tubes (universal white L 25, 36 W), installed above the water bath in a distance of about 35 cm from the test flasks.
The test duration was 72 hours.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: (3.1 - 41.0)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL(0.64 - 1.55)

Details on results:

The test substance had a statistically significantly inhibitory effect on the biomass of Scenedesmus subspicaii.is after the exposure period of 72 hours first at the mean measured concentration of 0.28 µg test substance/I (results of a Dunnett-test, one-sided, a = 0.05). However, the mean growth rate of the algae after 72 hours was statistically significantly reduced first at the next higher test concentration of in the mean 1.0 µg/I. Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects after the exposure period of 72 hours).
The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) was determined at the concentration of 0.28 µg test substance/I, since up to and including this test concentration the mean growth rate of the algae after the 72 hours exposure period was statistically not significantly lower than in the control.
At the microscopic examination of the shape of the algal cells after 72 hours test period no difference was observed between the algae growing in the undiluted filtrate and the algal cells in the control. Thus, the shape of the algal cells growing up to this test concentration of 1.0 g/1 was obviously not affected.
In the control the cell density has increased from nominal N = Ix io cells/mI at the start of the test (0 hours) to N = 123,00 x104 cells/mi (mean value) after 72 hours by a factor of 123 (Table I). Thus, the algal growth in the control was sufficiently high under the test conditions.
At the start of the test, the pH-values in the test media ranged from pH 7.9 to 8.1, at the end of the test pH-values were measured between pH 10.0 and 10.3 (Table 6). This increase of the pH during the test was obviously caused by the C02-consumption of the algae due to their rapid growth respectively their high densities (although the test media have been intensively stirred).
No remarkable observations were made concerning the behaviour of the test substance in the filtrate and in all dilutions of the filtrate.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

standard statistical methods were employed

None

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the EC50 values calculated for biomass and growth rate after 72 hours were:
EbC50: 0.90 µg/l (95% CL: 0.64 - 1.55) and ErC50: 6.7 µg/l (extrapolated); 95% CL: 3.1-41.0)

Executive summary:

The influence of the test substance MORTRACE SB CONCENTRATE on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992, and the OECD Guideline No. 201, adopted June 7, 1984. The test was performed in compliance with Good Laboratory Practice Regulations.

Due to the very low water solubility of the test substance a supersaturated stock suspension of the test substance with a nominal concentration of 100 mg/I was continuously stirred at room temperature in the dark over 24 hours. After 24 hours the stock suspension was filtered. The undiluted filtrate with the maximum concentration of dissolved respectively very fine dispersed test substance was used as the highest concentrated test medium. Additionally, the dilutions 1:100, 1:32, 1:10 and 1:3.2 were tested.

The analytically determined test substance concentrations in the freshly prepared test medium of the highest test concentration (the undiluted filtrate) amounted to 1.9 pig/I. At the lower concentrated test media lower test substance concentrations were measured according to the dilution steps down to 0.025 jig/I. However, the test substance was not stable under the test conditions during the test period. Therefore, all biological results are related to the mean measured test substance concentrations.

The 72-hour LOEC (lowest concentration tested with toxic effects after the exposure period of 72 hours) was determined at the mean measured concentration of 1.0 pig test substance/I. The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) was determined at 0,28 pig test substance/I, since up to and including this test concentration the mean growth rate of the algae was statistically not significantly lower than in the control.

Under the conditions of the study, the EC50 values calculated for biomass and growth rate after 72 hours were:

EbC50: 0.90 µg/l (95% CL: 0.64 - 1.55) and ErC50: 6.7 µg/l (extrapolated); 95% CL: 3.1-41.0)

Description of key information

In a growth inhibition test with Scenedesmus subspicatus, the 72-hour ErC50 for Mortrace SB Conc. was 6.7 μg/L.  The 72-hour NOEC based on growth rate was determined to be 0.28 μg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.7 µg/L

EC10 or NOEC for freshwater algae:

0.28 µg/L

Additional information

One static toxicity study with the green algae,Scenedesmus subspicatus,was available for review and was found to be of good quality and reliable for use in the risk assessment process (Klimisch score = 2). The Klimisch score for this study was 2 due to the instability of the test material in this study type. This study measured both growth rate (r) and biomass (b) as indicators of growth inhibition over 72 hours of exposure to Mortrace SB Conc.  A dilution series based on an initial loading rate of 100 mg/L was evaluated in this study, and the actual concentrations of Mortrace SB Conc. were evaluated both at the initial time of the experiment and at test termination, thus mean measured concentrations were available in the algal growth inhibition test. At the termination of 72 hours, a 10.9% growth inhibition was noted at the highest nominal loading rate of 100 mg/L, which equated to a mean measured concentration of 1.0 μg/L. To calculate the EC50, an extrapolation of the growth rate data was performed, and the E_rC50 value was estimated to be 6.7 μg Mortrace SB Conc/L. The 72-hour NOEC withS. subspicatuswas 0.28 μg Mortrace SB Conc/L based on mean measured concentrations.