Esterification products of salicylic acid and C13-(branched) alcohols

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:

18 July 2011 to 19 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tridecyl 2-hydroxybenzoate

EC Number:

947-975-7

Molecular formula:

C20H32O3

IUPAC Name:

tridecyl 2-hydroxybenzoate

Specific details on test material used for the study:

Identification Alcohol C12-13, branched and linear, salicylate
Molecular formula C6H4(OH)C(=O)-O-CH2-i-CnH2n+1 whereas n=11 or 12
Molecular weight 320.47
CAS Number 19666-16-1
Description Clear colourless liquid
Batch 110119
Purity 95.2% weight
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 19 January 2013 (taken from label)

Stability in water: Not indicated
Solubility in water: Negligible

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface).

The batch of Alcohol C12-13, branched and linear, salicylate tested was a clear colourless liquid with a purity of 95.2% weight and the substance was not completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 2-day period of magnetic stirring to ensure maximum dissolution. The obtained mixture was allowed to settle for a period of 24 hours. Subsequently, the Water Soluble Fraction (WSF) was siphoned off through a glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. Besides the undiluted WSF which was colourless and slightly hazy, the final test solutions were clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

Source In-house laboratory culture.
Reason for selection This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/l
K2HPO4.3H2O 52 mg/l
MgSO4.7H2O 75 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.2H2O 35 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
(NH4)6Mo7O24.4H2O 0.018 mg/l

Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 24 °C

pH:

7.9 - 8.2

Nominal and measured concentrations:

Nominal: 100 mg/l [Loading rate]
Measured mean: 0.59 mg/l

Details on test conditions:

Combined limit / range-finding test

Test concentrations

Alcohol C12-13, branched and linear, salicylate
Solutions containing 0.1, 1.0, 10 and 100% of the WSF prepared at a loading rate of 100 mg/l.

Controls
Test medium without test substance or other additives

Replicates
6 replicates each of the control and the highest concentration, 3 replicates of the remaining test concentrations, 1 replicate of the each test concentration without algae, 1 or 2 replicates of each test concentration for sampling purposes.

Test procedures and conditions

Test duration 72 hours

Test type Static

Test vessels 100 ml, all-glass, containing 50 ml of test solution

Medium M2

Cell density An initial cell density of 1 x 104 cells/ml.

Illumination
Continuously using TLD-lamps of the type „Cool-white‟ of 30 Watt, with a light intensity within the range of 80 to 90 E.m-2.s-1.

Incubation
Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Sampling for analysis of test concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (APPENDIX 5).

Frequency at t=0 h, t=24 h and t=72 h

Volume 2 ml

Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, singular reserve samples of 2 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements
pH
At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.

Temperature of medium
Continuously in a temperature control vessel.

Appearance of the cells
At the end of the combined limit/range-finding test microscopic observations were performed on every test concentration to check for any abnormal appearance of the algae.

Recording of cell densities
The highest test concentration was slightly hazy and contained small particles that could disturbed spectrophotometric measurement. Therefore algal density was determined in all groups by use of a microscope and a counting chamber throughout the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition were beyond the range tested, i.e. exceeded a TWA concentration of 0.59 mg/l present in the solution prepared at a loading rate of 100 mg/l.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Measured test substance concentrations
The results of analysis of the samples taken during the combined limit/range-finding test are described in Table 2 (of the analytical report).

Samples taken from the undiluted WSF (the highest test concentration) were analysed. At the start of the test, the actual test concentration was 2.9 mg/l. After 24 hours of exposure the measured concentration was at the level of 14% of initial and decreased further to 9.1% of initial at the end of the test. The Time Weight Average concentration was calculated to be 0.59 mg/l.

The measured concentration in the undiluted WSF incubated without algae was also 2.9 mg/l at the start of the test. The measured concentration also decreased to 8.0% of initial at the end of the test it was quite stable during the first 24 hours of exposure (81% of initial).

Mean cell densities
Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrations of Alcohol C12-13, branched and linear, salicylate. The respective growth curves are shown in Figure 1 (attached) (see APPENDIX 1 for the cell densities per replicate).


Table 1
Exposure time (hours)
Test substance* % WSF prep. at 100 mg/l 0 24 48 72
Control 1 7.1 27.5 149.9
0.1 1 5.8 28.4 148
1 1 5.6 30.6 171.3
10 1 4.8 29.3 166.7
100 (0.59) 1 5.3 29.5 165.5

* - Alcohol C12-13, branched and linear, salicylate
() – between brackets: TWA concentration

Reduction of growth rate and inhibition of yield

Table 2 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition. The calculation of the percentages of growth rate reduction at different time intervals are shown in Table 3 (see APPENDIX 1 for the values of growth rate and yield per replicate).

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Table 2

Mean growth rate Yield (0 - 72 h)
Test substance* % WSF prep. at 100 mg/l µ (0-72 h) Reduction (%)1 x 10^4 cells/ml Inhibition1 (%)
Control 0.06955 148.92
0.1 0.0694 0.2 147 1.3
1 0.07141 -2.7 170.33 -14.4
10 0.07105 -2.2 165.67 -11.2
100 (0.59) 0.07095 -2 164.5 -10.5

* - Alcohol C12-13, branched and linear, salicylate
1. Negative numbers indicate stimulation of growth or increase of yield.
() – between brackets: TWA concentration

Table 3

Mean growth rate Mean growth rate Mean growth rate
Test substance* % WSF prep. at 100 mg/l µ (0-24 h) Reduction (%)1 µ (24-48 h) Reduction (%)1 µ (48-72 h) Reduction (%)1
Control 0.08178 0.05621 0.07066
0.1 0.07286 10.9 0.06642 -18.1 0.06892 2.5
1 0.07132 12.8 0.07106 -26.4 0.07186 -1.7
10 0.0648 20.8 0.0758 -34.8 0.07255 -2.7
100 (0.59) 0.06965 14.8 0.0713 -26.8 0.07192 -1.8

* - Alcohol C12-13, branched and linear, salicylate
1. Negative numbers indicate stimulation of growth or increase of yield.
() – between brackets: TWA concentration

Determination of effect concentrations
Table 4 shows the effect parameters based on Time Weight Average concentrations.

Table 4 Effect parameters

Parameter TWA1 concentration Alcohol C12-13, branched and linear, salicylate (mg/l)
NOERC 0.59
72h-ERC10 >0.59
72h-ERC50 >0.59
NOEYC 0.59
72h-EYC10 >0.59
72h-EYC50 >0.59
1. Time Weight Average

Experimental conditions
Table 5 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 22.0°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.5 and 23.6°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

Table 5 pH levels
Test substance*
TWA1 conc.
(mg/l) Exposure time (hours)
0 72
control 8.1 8.1
0.59 8.2 7.9
* - Alcohol C12-13, branched and linear, salicylate
1. Time Weight Average concentration

Results with reference substance (positive control):

See attached

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of Alcohol C12-13, branched and linear, salicylate tested. This indicates that in the range of water solubility the test substance was not toxic under the test conditions.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition were beyond the range tested, i.e. exceeded a TWA concentration of 0.59 mg/l present in the solution prepared at a loading rate of 100 mg/l.

The 72-h EL50 for Alcohol C12-13, branched and linear, salicylate the test item to Pseudokirchneriella subcapitata based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

The NOEC for growth rate reduction and yield inhibition was 0.59 mg/l. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Alcohol C12-13, branched and linear, salicylate.

The study procedures described in this report were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000.

The batch of Alcohol C12-13, branched and linear, salicylate tested was a clear colourless liquid with a purity of 95.2% weight and the substance was not completely soluble in test medium at the concentrations tested.

A combined limit/range finding test was performed. Preparation of test solutions started with a loading rate of 100 mg/l applying a 2-day period of magnetic stirring to ensure maximum dissolution. The obtained mixture was allowed to settle for a period of 24 hours. Subsequently, the Water Soluble Fraction (WSF) was siphoned off through glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. Besides the undiluted WSF which was colourless and slightly hazy, the final test solutions were clear and colourless.

Six exponentially growing algal cultures per group were exposed to a control and the undiluted WSF in the limit test. In addition, three replicates per group were exposed to solutions containing 0.1, 1.0 and 10% of the WSF in the range-finding test. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at test initiation, 24 hours later and at the end of the test.

Samples taken from the undiluted WSF were analysed. At the start of the test, the actual test concentration was 2.9 mg/l. After 24 hours of exposure the measured concentration was at the level of 14% of initial and decreased further to 9.1% of initial at the end of the test. The Time Weight Average concentration was calculated to be 0.59 mg/l.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of Alcohol C12-13, branched and linear, salicylate tested. This indicates that in the range of water solubility the test substance was not toxic under the test conditions.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition were beyond the range tested, i.e. exceeded a TWA concentration of 0.59 mg/l present in the solution prepared at a loading rate of 100 mg/l.

The 72-h EL50 for Alcohol C12-13, branched and linear, salicylate the test item to Pseudokirchneriella subcapitata based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

The NOEC for growth rate reduction and yield inhibition was 0.59 mg/l. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.