N-(dodecylphenyl)naphthalen-1-amine

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:

13 October 2000 to 17 November 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the centrifuged and uncentrifuged test samples were verified by chemical analysis at 0 and 72 hours.

Vehicle:

yes

Remarks:

dimethylforinamide

Details on test solutions:

For the purpose of the definitive study, the test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (100 mg) was dissolved in dimethylforinamide and the volume adjusted to 10 ml to give a 100 mg/10 ml solvent stock solution. Ail aliquot (200 μl) of this solvent stock solution was dispersed in 2 litres of algal suspension to give the required 1.0 mg/l test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276120. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.
Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.
Culture Medium
The culture medium used for both the range-finding and definitive studies was the same as that used to maintain the stock culture.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified

Test temperature:

21 ± 1 °C

pH:

7.6 - 10.5

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The range-finding study was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 0.10 and 1.0 mg/l for a period of 72 hours.
Based on the result of the range-finding study a "limit test" was conducted for the definitive study at a test concentration of 1.0 mg/l to confirm that at the highest attainable test concentration of 1.0 mg/l.
The time-weighted mean measured test concentrations of the centrifuged samples using the mean of the measured test concentrations of replicates R1 - R3 pooled and replicates R4 - R6 pooled were calculated (see Any other information for full details).

Details on test conditions:

Procedure
Range-finding study
The test concentration to be used in the definitive study was determined by a preliminary range finding study. During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. However, at a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (SafePharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.
Based on this information it was considered appropriate to conduct the range-finding study using a homogenous dispersion of the test material to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration. Therefore the range-finding study was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 0.10 and 1.0 mg/l for a period of 72 hours.
The study was conducted in 250 ml glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
The test material was dissolved in dimethylformamide.
An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 100 mg/10 ml solvent stock solution from which a dilution was made to give a further solvent stock solution of 10 mg/10 ml. An aliquot (20 μl) of each of the solvent stock solutions was separately dispersed in 200 ml of algal suspension to give the required test concentrations of 0.10 and 1.0 mg/l.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl/l of dimethylformarnide.
At the start of the range-finding study a sample of each test and control culture was removed and the cell density determined using a coulterm Multisizer II Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (Gallenkamp INR - 40 1-0 10 W) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a coulter Multisizer II Particle Counter.

Definitive study
Based on the result of the range-finding study a "limit test" was conducted for the definitive study at a test concentration of 1.0 mg/l to confirm that at the highest attainable test concentration of 1.0 mg/l, no effect on algal growth was observed.

Exposure conditions
As in the range-finding study 250 ml glass conical flasks were used.
Six flasks each containing 100 ml of solution were prepared for the treatment group and three flasks each for the control and solvent control group.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl/l of dimethylformamide.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 7.87e+5 cells per ml. This suspension was diluted to a cell density of 1.17e+4 cells per ml prior to use.
The flasks were plugged with polyurethane foam bungs and incubated (Gallenkamp INR-401-010W) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a coulter Multisizer II Particle Counter.

Physico-chemical measurements
The pH of each control, solvent control and test flask was determined at initiation of the study and after 72-hours exposure. The temperature within the incubator was recorded daily.

Verification of test concentrations
Two samples of the solvent control and the 0.10 mg/l test groups (replicates R1 – R3 and R4 – R6 pooled) were taken at 0 and 72 hours. One sample was analysed unfiltered and one sample after centrifugation (17,000 G). Further samples (in duplicate) were taken at 0 hours and stored frozen (approximately -20°C) for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Study
During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. However, at a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (SafePharm Laboratories Project Number: 445/283) showed a water solubility, value of 1.96 mg/l.
Based on this information it was considered appropriate to conduct the range-finding study using a homogenous dispersion of the test material to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration. Therefore, the range-finding study was conducted using a series of nominal test concentrations of 0.10 and 1.0 mg/l.
The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/l.
Based on this information a single test concentration of six replicates, of 1.0 mg/l was selected for the definitive study. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration of 1.0 mg/l no effect on growth was observed.

Definitive Study
Growth data
From the data given, it is clear that neither the growth (r) or the biomass (b) of Scenedesmus subspicatus (CCAP 276120) were affected by the presence of the test material over the 72-hour exposure period.
The EC50 value with respect to biomass, EbC50 (72 h), was determined by inspection of the area under the growth curve data after 72 hours.
The EC50 value with respect to growth rate, ErC50 (0 - 72 h), was determined by inspection of the growth rates for the period 0 - 72 hours.
Accordingly the following results were determined from the data:
EbC50 (72 h) : >1.0 mg/l
ErC50 (0 – 72 h) : >1.0 mg/l
where EbC50 is the test concentration that reduced biomass by 50% and ErC50 is the test concentration that reduced specific growth rate by 50%.
Statistical analysis of the area under the growth curve data was carried out for the solvent control and 0.10 mg/l test group using a Students t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P≥0.05) between the solvent control and 1.0 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) was 1.0 mg/l.
The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.
The following data show that the cell concentration of the control cultures increased by a factor of 161 and the cell concentration of the solvent control cultures increased by a factor of 150 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:
Mean cell density of control at 0 hours: 1.08e+4cells per ml
Mean cell density of control at 72 hours: 1.744+6 cells per ml
Mean cell density of solvent control at 0 hours: 1.054+4 cells per ml
Mean cell density of solvent control at 72 hours: 1.57e+6 cell per ml

Observations
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Physico-chemical measurements
Temperature was maintained at 24 ± 1 °C throughout the study.
The pH values of the control cultures were observed to increase from pH 7.6 at 0 hours to pH 10.4 - 10.5 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Verification of test concentrations
During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. However, at a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Therefore, it was considered appropriate to conduct the test using a test concentration of 1.0 mg/l to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration. Furthermore, a study, The Determination of the General Physico-Chemical properties of the test material (SafePharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.
Recovery analyses for the Acute Toxicity to Rainbow Trout study (SafePharm Laboratories Project Number 445/299) were performed on test samples prepared at nominal concentrations of 0.10 and 1.0 mg/l in dechlorinated laboratory tap water. Samples were taken and analysed after both single and double 0.2 μm filtration. The results obtained indicated that the test material adsorbed to the filter matrix.
Given these results it was considered appropriate to conduct the algal inhibition recovery and stability analyses on centrifuged and un-centrifuged samples alone. Samples were prepared at a concentration of 1.0 mg/l and analysed untreated alone and with algal cells and after centrifugation, alone and with algal cells (17,000 G). These results indicated that a proportion of the test material was present as a dispersion at the concentration of 1.0 mg/l.
Based on these results it was considered appropriate to analyse the samples taken from the definitive study with no pre-treatment and after 30 minutes centrifugation (17,000 G). The results of the centrifuged samples giving a measure of the concentration of test material in solution and hence bioavailable to the test organisms.
Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 92% to 101% of nominal for the un-centrifuged samples and from 53% to 70% of nominal for the centrifuged samples. Analysis of the test solutions at 72 hours showed the measured test concentrations of both the un-centrifuged and centrifuged samples to be below the limit of quantitation of the analytical method employed. Given that the pre-study stability analyses conducted indicated that the test material was stable in culture medium over the study period this decline was considered to be due to adsorption to algal cells. Although the pre-study recovery analyses conducted in the presence of algal cells indicated no immediate adsorption to algal cells this does not preclude long term adsorption. Adsorption was not a factor in the stability analysis since no algal cells were present.
Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged samples in order to give a "worst case" analysis of the data. Following the current advice of the competent authorities in the EC, in cases where the measured concentration was less than the LOQ (Limit of quantitation) of the analytical method, a concentration of half of the LOQ (i.e. 0.033 mg/l) was used for the purposes of calculating the time-weighted mean measured test concentrations.
The following results are based on the time-weighted mean measured test concentrations of the centrifuged samples:
EbC50 (72h) : > 0.20 mg/l
ErC50 (0-72h) : > 0.20 mg/l
No Observed Effect Concentration (NOEC) = 0.20 mg/l.
The use of time-weighted mean measured test concentrations had a significant effect on the results of the study. This was considered to be due to the dissolved portion of the test material being significantly lower than the nominal test concentration, and the adsorption of the test material to algal cells over the study period further reducing the amount of dissolved test material present after 72 hours.

Results with reference substance (positive control):

Positive control not performed as part of the study.

Reported statistics and error estimates:

A Students t-test incorporating Bartlett's test for homogeneity of variance was carried out on the area under the growth curve data at 72 hours for the solvent control and the 0.10 mg/l test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Cell Densities and Percentage Inhibition of Growth from the Range-finding Study

Nominal Concentration (mg/l)		Cell Densities* (cells per ml)
		0 Hours	72 Hours	% Inhibition (area under curve at 72 h)
Control	R1 R2 Mean	1.05 x 10^4 1.09 x 10^4 1.07 x 10^4	1.65 x 10^6 1.47 x 10^6 1.56 x 10^6	-
Solvent Control	R1 R2 Mean	1.13 x 10^4 1.08 x 10^4 1.11 x 10^4	1.41 x 10^6 1.48 x 10^6 1.44 x 10^6	-
0.10	R1 R2 Mean	1.10 x 10^4 1.20 x 10^4 1.15 x 10^4	1.59 x 10^6 1.31 x 10^6 1.45 x 10^6	0
1.0	R1 R2 Mean	1.06 x 10^4 1.10 x 10^4 1.08 x 10^4	1.24 x 10^6 1.47 x 10^6 1.35 x 10^6	6

* Cell densities represent the mean number of cells per ml calculated from the mean cell counts from 3 counts for each of the replicate flasks.

R₁– R₂= Replicate 1 to 2

 

Cell Densities and pH Values in the Definitive Study

Nominal Concentration (mg/l)		pH	Cell Densities* (cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1 R2 R3 Mean	7.6 7.6 7.6	1.12 x 10^4 1.09 x 10^4 1.02 x 10^4 1.08 x 10^4	8.59 x 10^4 8.36 x 10^4 8.73 x 10^4 8.56 x 10^4	4.61 x 10^5 4.82 x 10^5 6.29 x 10^5 5.24 x 10^5	1.88 x 10^6 1.57 x 10^6 1.76 x 10^6 1.74 x 10^6	10.5 10.5 10.4
Solvent Control	R1 R2 R3 Mean	7.5 7.5 7.5	1.01 x 10^4 1.04 x 10^4 1.10 x 10^4 1.05 x 10^4	8.32 x 10^4 7.90 x 10^4 7.68 x 10^4 7.97 x 10^4	5.22 x 10^5 5.30 x 10^5 5.57 x 10^5 5.36 x 10^5	1.60 x 10^6 1.40 x 10^6 1.71 x 10^6 1.57 x 10^6	10.5 10.4 10.4
1.0	R1 R2 R3 R4 R5 R6 Mean	7.3 7.3 7.3 7.3 7.3 7.3	1.07 x 10^4 1.09 x 10^4 1.03 x 10^4 1.07 x 10^4 1.15 x 10^4 1.29 x 10^4 1.12 x 10^4	8.16 x 10^4 8.57 x 10^4 8.63 x 10^4 8.65 x 10^4 8.73 x 10^4 8.69 x 10^4 8.57 x 10^4	5.13 x 10^5 5.15 x 10^5 5.07 x 10^5 5.02 x 10^5 5.02 x 10^5 5.20 x 10^5 5.10 x 10^5	1.63 x 10^6 1.58 x 10^6 1.67 x 10^6 1.76 x 10^6 1.74 x 10^6 1.36 x 10^6 1.62 x 10^6	10.1 10.2 10.1 10.2 10.0 10.0

* Cell densities represent the mean number of cells per ml calculated from the mean cell counts from 3 counts for each of the replicate flasks.

R₁– R₆= Replicate 1 to 6

 

Inhibition of Growth Rate and Biomass

Nominal Concentration (mg/l)	Area Under Curve at 72 h	% Inhibition	Growth Rate (0-72 h)	% Inhibition
Control	3.48 x 10^7	-	0.071	-
Solvent Control	3.30 x 10^7	-	0.070	-
1.0	3.31 x 10^7	0	0.069	1

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 1.0 mg/l. Correspondingly the No Observed Effect Concentration was 1.0 mg/l.
Based on the time-weighted mean measured test concentrations the EC50 values were estimated to be greater than 0.20 mg/l. Correspondingly the No Observed Effect Concentration was equal to 0.20 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Following a preliminary range-finding study, Scenedesmus subspicatus as exposed to an aqueous solution of the test material at a concentration of 1.0 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer II Particle Counter.

 

Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 1.0 mg/l and correspondingly the No Observed Effect Concentration was 1.0 mg/l.

 

The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Furthermore, a study, The Determination of the General Physico-Chemical properties of the test material (Safepharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.

 

Recovery analyses for the Acute toxicity to Rainbow Trout study (Safepharm Laboratories Project Number 445/399) were performed on test samples prepared at nominal concentrations of 0.10 and 1.0 mg/l in dechlorinated laboratory tap water. Samples were taken and analysed after both single and double 0.2 μm filtration. The results obtained indicated that the test material adsorbed to the filter matrix.

 

Given these results it was considered appropriate to conduct the algal inhibition recovery and stability analyses on centrifuged and un-centrifuged samples alone. Samples were prepared at a concentration of 1.0 mg/l and analysed untreated alone and with algal cells and after centrifugation, alone and with algal cells (17,000 G). These results indicated that a proportion of the test material was present as a dispersion at the concentration of 1.0 mg/l.

 

Based on these results it was considered appropriate to analyse the samples taken from the definitive study with no pre-treatment and after 30 minutes centrifugation (17,000 G). The results of the centrifuged samples giving a measure of the concentration of test material in solution and hence bioavailable to the test organisms.

 

Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 92% to 101% of nominal for the un-centrifuged samples and from 53% to 70% of nominal for the centrifuged samples. Analysis of the test solutions at 72 hours showed the measured test concentrations of both the un-centrifuged and centrifuged samples to be below the limit of quantitation of the analytical method employed. This decline in measured test concentrations was considered to be due to adsorption to algal cells.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged media in order to give a "worst case" analysis of the data. The EC50 values based on the time-weighted mean measured test concentrations were greater than 0.20 mg/l and correspondingly the No Observed Effect Concentration was 0.20 mg/l.

Description of key information

GLP accredited laboratory study performed in accordance with OECD Guideline 201 and EU Method C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.2 mg/L

EC10 or NOEC for freshwater algae:

0.2 mg/L

Additional information

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus.

 

Following a preliminary range-finding study, Scenedesmus subspicatus as exposed to an aqueous solution of the test material at a concentration of 1.0 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 1.0 mg/l and correspondingly the No Observed Effect Concentration was 1.0 mg/l.

 

The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Furthermore, a study,showed a water solubility value of 1.96 mg/l.

 

Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 92% to 101% of nominal for the un-centrifuged samples and from 53% to 70% of nominal for the centrifuged samples. Analysis of the test solutions at 72 hours showed the measured test concentrations of both the un-centrifuged and centrifuged samples to be below the limit of quantitation of the analytical method employed. This decline in measured test concentrations was considered to be due to adsorption to algal cells.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged media in order to give a "worst case" analysis of the data. The EC50 values based on the time-weighted mean measured test concentrations were greater than 0.20 mg/l and correspondingly the No Observed Effect Concentration was 0.20 mg/l.