4-hydroxy-3,5-dimethoxybenzonitrile

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-27 to 2008-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Tan, powder
- Analytical purity: 99.5%
- Storage condition of test material: ambient

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

Test substance preparation:
-Test material was determined to be slightly soluble in water. For all test concentrations prepared, a water-soluble fraction was prepared by adding the required nominal amount of test substance in moderately hard synthetic freshwater, and mixing the solution for 20-24 hours. After mixing, the undissolved test substance was separated from the solution, and only this water-soluble fraction was used for testing.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): STILLMEADOW, Inc. cultures
- Age of inoculum (at test initiation): Mass culture initiated 3-7 ±1 days prior to dosing
- Density: Initial cell density was 10,000 cells/mL
- Culturing media and conditions (same as test or not): Algal stock culture medium (Woods Hole) with EDTA up to 0.3 mg/L. 3 days prior to testing, 1L of autoclaved algal stock culture medium was inoculated. This mass culture was then placed in a ~25°C environmental chamber and maintained on continuous aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Moderately hard synthetic freshwater

Test temperature:

22-24°C

pH:

Terminal pH of test concentrations were (avg taken from triplicate beakers):
1 mg/L - 8.4
2 mg/L - 8.7
4 mg/L - 8.3
8 mg/L -7.9
16 mg/ L - 7.8
Positive control - 7.1

Dissolved oxygen:

Test containers received constant aeration.

Salinity:

NA

Nominal and measured concentrations:

1, 2, 4, 8, and 16 mg/L (nominal)

Details on test conditions:

Test chambers: 250-mL sterile Erlenmeyer flasks with stoppers and aeration tubing; filled with 100 mL of appropriate solution
Test containers received constant aeration.
Algal stock medium with EDTA up to 0.3 mg/L.
Temp: 22-24°C

Range finding test:
Five concentrations used: 1, 10, 100, 500, 1000 mg/L, One flask for each conc. Algae introduced to each conc. at initial cell density of 10,000 cells/mL. At 96 hours, the terminal density was measured.

Definitive test: 1, 2, 4, 8, 16 mg/L used. Three flasks per conc and control. The flasks were set on a shaker table for constant agitation at ~100 cycles/minute. At 24, 48, 72, and 96 hours after dosing, observations of inhibition were made for each container by measuring the mean cell density with a haemocytometer. The temperature was recorded continuously, and the pH of each container was recorded at study termination.

Reference substance (positive control):

yes

Remarks:

10 mg/L zinc chloride

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

3.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CL of 0.95 - 16.7 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Results with reference substance (positive control):

72-hour interim control density and growth rate increased by a factor of at least 16.

Reported statistics and error estimates:

The NOEC and IC50 were determined by a commercially available statistical program (ToxCalc Version 5.0TM). Several models are available for IC50 determination: Probit, Trimmed Spearman-Karber, and Linear Interpolation. The appropriate model was selected for the determination in this case.

Validity criteria fulfilled:

yes

Conclusions:

The test concentration of Mediator SNP, (4-hydroxy-3,5-dimethoxybenzonitrile) that resulted in 50% inhibition of Selenastrum capricornutum in freshwater was 3.99 mg/L. The NOEC (no observed effect concentration) was <1 mg/L.

Executive summary:

This study was conducted to determine the toxic effects of the test substance 4-hydroxy-3,5-dimethoxybenzonitrile on the growth of the freshwater algae Selenastrum capricornutum in a 96-hour test in accordance with OECD Guideline 201. The test concentration of 4-hydroxy-3,5-dimethoxybenzonitrile that resulted in 50% inhibition (IC50) was determined to be 3.99 mg/L with 95% confidence limits of 0.95 -16.73 mg/L. The NOEC (No Observed Effect Concentration) was determined to be <1 mg/L. Results presented are based on nominal concentrations. This classifies the test material as Category Acute 1: "very toxic to aquatic life".

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-09-08 to 2009-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

The four ingredients, monosodium phosphate, adipic acid, laccase granule and 4-hydroxy-3,5-dimethoxybenzonitrile, were dissolved in culture medium with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 200 mg mediator laccase blend stock solution. A series of dilutions was made from the 200 mg/L stock solution to give further stock solutions of 150, 75, 25 and 12.5 mg/L. An aliquot (500 mL) of each of the 12.5, 25, 75 and 150 mg/L stock solutions was separately inoculated with algal suspension (10 mL) to give the required test concentrations of 12.5, 25, 75 and 150 mg/L.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: formerly known as Scenedesmus subspicatus
-strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and constant illumination at 21 ± 1°C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10000 - 100000 cells/mL.
The culture medium used for the definitive test was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 12.5, 25 and 75 mg/L; however few intact cells were observed to be present in the test cultures at 150 mg/L.

Test temperature:

24 ± 1°C

pH:

12.5 mg/L: start - 7; end - 7.7
25 mg/L: start - 6.8; end - 7.5
75 mg/L: start - 6.1; end - 6.9
150 mg/L: start - 5.4; end - 5.2
control: start - 7.4; end - 7.9
Avg. values reported here.

Nominal and measured concentrations:

Nominal concentrations at 12.5, 25, 75 and 150 mg/L.

Details on test conditions:

- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks, 100 mL of appropriate solution in the flask
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.
- Initial cells density: 4E3 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water
NaN03 25.5 mg/L
MgCh.6H2O 12.164 mg/L
CaCh.2H2O 4041 mg/L
MgS04.7H20 14.7 mg/L
K2HP04 1.044 mg/L
NaHC03 15.0 mg/L
H3B03 0.1855 mg/L
MnChAH20 00415 mg/L
ZnCh 0.00327 mg/L
FeCi).6H2O 0.159 mg/L
CoCh.6H20 0.00143 mg/L
Na2Mo04.2H20 0.00726 mg/L
CuCh.2H2O 0.000012 mg/L
Na2EDTA.2H20 0.30 mg/L
Na2Se03.5H20 0.000010 mg/L
pH adjusted to 7.5 ± 0.1 with 0.01 N NaOH or HCI.

-The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
-The temperature within the incubator was recorded daily.
OTHER TEST CONDITIONS
-continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density
- Determination of cell concentrations: cell densities determined using a Coulter@ Multisizer Particle Counter.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 100-120 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL = 55-84 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL = 68-96 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

ErC50 (0 - 72 h) of 0.57 mg/L; 95% confidence limits 0.48 - 0.66 mg/L, an EyC50 (0 - 72 h) of 0.32 mg/L; 95% confidence limits 0.29 - 0.35 mg/L,
and an EbC50 (0 - 72 h) of 0.31 mg/L; 95% confidence limits 0.28 - 0.35 mg/L.
The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L, respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L, respectively.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validity criteria fulfilled:

yes

Remarks:

The results from the positive control with potassium dichromate were within the normal range for this reference material.

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-hour period and gave an ErC50 (0 - 72 h) of 110 mg/L (95% confidence limits 100 - 120 mg/L), an EyC50 (0 - 72 h) of 68 mg/L (95% confidence limits 55 - 84 mg/L), and an EbC50 (0 - 72 h) of 81 mg/L (95% confidence limits 68 - 96 mg/L). The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 25 mg/L, and the No Observed Effect Concentration was 12.5 mg/L.

Executive summary:

The form in the supply chain for 4-hydroxy-3,5-dimethoxybenzonitrile is in conjunction with laccase enzyme, adipic acid, and monosodium phosphate, a ready-to-use product in the textile industry for denim bleaching applications (Tradename: PrimaGreen EcoFade LT100). This composition is referred to here as RTU blend. In order to examine the toxicity to aquatic algae of the ready-to-use commercial product, this study was conducted in accordance with OECD Guideline 201.

Desmodesmus subspicatus was exposed to an aqueous solution of the test material (RTU product) at concentrations of 12.5, 25, 75 and 150 mg/L 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.

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 110 mg/L; 95% confidence limits 100 - 120 mg/L. In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 68 mg/L; 95% confidence limits 55 - 84 mg/L. In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 81 mg/L; 95% confidence limits 68 - 96 mg/L.

The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 25 mg/L, and the No Observed Effect Concentration was 12.5 mg/L. Under GHS guidelines, this classifies the test material as "not acutely toxic to algae".

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-04-27 to 2011-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Tan, powder
- Analytical purity: 99.5%
- Storage condition of test material: ambient conditions in the dark.

Analytical monitoring:

yes

Details on sampling:

Samples of the test solution were collected at approximately 0 and 72 hours to measure concentrations of the test substance. At test initiation samples were collected for each treatment and control group prior to distribution into test chambers. At test termination, the biological replicates from each respective treatment and control group were pooled and then sampled. All samples were collected in glass vials and processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

A primary stock solution was prepared by dissolving 0.0151 g of syringonitrile (test article) in 1000 mL of freshw ter AAP algal medium to achieve a nominal concentration of 15 mg a.i./L. The solution was mixed by inversion and was sonicated for ten minutes. It was stirred with a stirbar and magnetic stir plate while all subsequent dilutions were made. The stock solution was proportionately diluted with freshwater AAP algal medium to prepare 5 additional test solutions at target nominal concentrations of 0.045, 0.14, 0.46, 1.5 and 4.7 mg a.i./L. All test solutions were mixed by inversion, and appeared clear and coloruless. The negative control solution consisted of freshwater AAP algal medium without the test substance.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): from University of Toronto Culture Collection, maintained in culture medium at Wildlife International, Ltd, Easton, Maryland, USA
- Age of inoculum (at test initiation): Algal cells used in this test were obtained from Wildlife International, Ltd, cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for the study were taken from a culture that had been transferred to fresh media three days prior to test initiation.
The cells were cultured and tested in freshwater AAP algal medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples of test solution were collected from each of the replicates per treatment and control group at the end of the test. They w ere examined microscopically for atypical cell morphology. Cells in the replicate test chambers also were assessed for aggregations of flocculations of cells, and adherence of the cells to the test chamber.

Hardness:

N/A

Test temperature:

22C-26°C

pH:

Ranged from 7.2 to 7.4 at test initiation, and from 7.6 to 8.6 at test termination. The observed increase in pH is typical for tests conducted with P. subcapitata and is attributed to the photosynthetic activity of the algae.

Dissolved oxygen:

Test flasks received constant aeration.

Salinity:

N/A

Nominal and measured concentrations:

Nominal concentrations= 0.045, 0.14, 0.46, 1.5, 4.7, and 15 mg a.i./L.
Measured concentrations = 0.042, 0.14, 0.45, 1.5, 4.8 and 15 mg a.i./L.
Results of the study were based on nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks
- Type (delete if not applicable): closed; plugged with foam stoppers
- Material, size, headspace, fill volume: each flask contained 100 mL of test medium.
- Aeration: all flasks were placed on a mechanical shaker at 100 rpm throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 10000 cells/mL
- Control end cells density: The mean cell density in the negative control flasks increased by a factor of 291 after three days.
- No. of organisms per vessel: 10000 cells/mL x 100 mL = 1000000 cells of P. subcapitata
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): Negative ctrl (medium with no test substance) = 6

GROWTH MEDIUM
- Standard medium used: freshwater AAP algal medium
- Detailed composition if non-standard medium was used: Medium constituents are also listed in the report.

Component: Nominal concentration
MgCl2 6H2O: 12.164 mg/L
CaCl2 2H2O: 4.410 mg/L
H3BO3: 0.1855 mg/L
MnCl2 4H20: 0.4154 mg/L
ZnCl2: 3.27 µg/L
FeCl3 6H2O: 0.1598 mg/L
CoCl2 6H2O: 1.428ug/L
Na2MoO4 2H2O: 7.26 µg/L
CuCl2 2H2O: 0.012 µg/L
Na2 EDTA 2H2O: 0.300 mg/L
NaNO3: 25.50 mg/L
MgSO4 7H2O: 14.70 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.00 mg/L

TEST MEDIUM -- same as Growth medium

WATER PARAMETERS
Analyses of Pesticides, Organics, and Metals in Wildlife International, Ltd. Well water
Component, Measured Concentration (µg/L)
Aldrin <0.0098
Alpha BHC <0.0098
Alpha Chlordane <0.0098
Beta BHC <0.0098
Bolstar <2.0
Chlordane <0.49
Coumaphos <2.9
Delta BHC <0.0098
Demeton-O <2.0
Demeton-S <2.9
Diazinon <5.9
Dichlorvos <2.9
Dieldrin <0.020
Disulfoton <2.0
Dursban(Chlorpyrifos) <2.9
Endosulfan I<0.0098
Endosulfan II<0.020
Endosulfan sulfate <0.020
Endrin<0.020
Endrin Aldehyde <0.098
Endrin Ketone <0.020
EPN <2.0
Ethion <2.0
Ethoprop <2.9
Ethyl Parathion <2.0
Famphur <2.9
Fensulfothion <8.8
Fenthion <2.0
Gamma BHC-Lindane<0.0098
Gamma Chlordane <0.020
Guthion (Azinphos-methyl) <5.9
HCB <0.0098
Heptachlor <0.0098
Heptachlor Epoxide <0.0098
Kepone <0.20
Malathion <2.9
Merphos <2.0
Methoxychlor <0.098
Methyl Parathion<2.0
Mevinphos <4.9
Mirex <0.25
Naled <5.9
o,p-DDD <0.020
o,p-DDE <0.74
o,p-DDT <0.020
p,p-DDD<0.020
p,p-DDE <0.020
p,p -DDT <0.020
Phorate <2.0
Ronnel <2.0
Stirophos <2.0
Telodrin <0.0098
Tokuthion <2.0
Toxaphene <2.9
Trichloronate <2.0
Trithion <2.0

Component, measured concentration (mg/L)
Aluminum <0.200
Antimony <0.0200
Arsenic <0.0200
Barium <0.0050
Beryllium <0.0050
Bromide <2.5
Cadmium <0.0050
Calcium 35.1
Chloride 3.5
Chromium <0.0150
Cobalt <0.0050
Copper <0.0100
Fluoride<0.50
Iron <0.200
Lead <0.0150
Magnesium 13.0
Manganese <0.0050
Mercury <0.00020
Nickel <0.0100
NItrate Nitrogen 0.55
Nitrite Nitrogen <0.50
Potassium 6.77
Selenium <0.0200
SIlver <0.0050
Sodium 18.5
Sulfate 5.6
Thallium <0.0300
Vanadium <0.0050
Zinc <0.0200

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH was adjusted to 7.5 using 10% hydrochloric acid.
- Photoperiod: The algae were held under continuous cool-white fluorescent lighting throughout the test.
- Light intensity and quality: The target intensity was 6000 lux ± 10%.
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Test medium samples were collected from each replicate of the treatment and control group for the determination of the cell densities at approximately 24-hour intervals during the 72-hour exposure. Cell counts were performed using an electronic particle counter.
- Chlorophyll measurement: N/A
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Range finding study performed. See below .
- Range finding study--yes. nominal test concentrations were chosen based upon the results of an exploratory range finding toxicity test.
- Test concentrations: Nominal test conc. were 0.045, 0.14, 0.46, 1.5, 4.7, and 15 mg a.i./L.
- Results used to determine the conditions for the definitive study: 72-hour range finding test with P. subcapitata (see below)
Nominal concentration (mg a.i /L)/ 72 - hour cell density(cells/mL)/ % inhibition vs. Control (%)
Negative control/ 1280000/ NA
0.01/1230000/ 4
0.1/1000000/ 22
1/ 948000/26
10/ 494000/61
100/117000/91
-appearance of test solutions: clear and colourless, with no precipitation noted.
Proposed concentrations for the definitive test:
Negative ctrl, 0.045, 0.14, 0.46, 1.5, 4.7 and 15 (mg a.i./L)

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell density

Remarks on result:

other: 95% CI = 6.3-8.0 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI = 6.3-8.0 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell density

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): P. subcapitata cell density was observed over the study period.
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: none
- Any stimulation of growth found in any treatment: 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

Mean Cell Density, Mean Yield and Percent Inhibition
Nominal Test Concentration (mg a.i./L)	24 Hours		48 Hours		72 Hours		72-Hour Yield^3
	Mean Cell Density^1(cells/mL)	% inhibition^1,2	Mean Cell Density^1(cells/mL)	% inhibition^1,2	Mean Cell Density^1(cells/mL)	% inhibition^1,2	Mean Yield^1,3(cells/mL)	% inhibition^1,2
Negative Control	91678	--	568247	--	2906211	--	2896211	--
0.045	91420	0	593936	-5	2807459	3	2797459	3
0.14	89254	3	587697	-3	2805628	3	2795628	3
0.46	94799	-3	580656	-2	3019979	-4	3009979	-4
1.5	86591	6	538665	5	2913629	0	2903629	0
4.7	72560	21	333061	41	1921625*	34	1911625*	34
15	72560	17	210029	63	608697*	79	598697*	79
^1Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly. ^2Percent inhibition was calculated relative to the negative control mean (n=6). ^3Yield was calculated at 72 hours as the final biomass (cell density) in the exposure period minus the initial biomass (10000 cells/mL). *Treatment group mean significantly different (Dunnett’s test, p<0.05) from the negative control mean. Only evaluated at 72 hours, per study protocol.

Mean Growth Rate (per hour) and Percent Inhibition
Nominal Test Concentration (mg a.i./L)	0-24 Hours		0-48 Hours		0-72 Hours
	Mean Growth Rate (days-1)^1(cells/mL)	% inhibition^1,2	Mean Growth Rate (days-1)^1(cells/mL)	% inhibition^1,2	Mean Growth Rate (days-1)^1(cells/mL)	% inhibition^1,2
Negative Control	0.0922	NA	0.0842	NA	0.0788	NA
0.045	0.0921	0	0.0849	-1	0.0783	1
0.14	0.0912	1	0.0848	-1	0.0783	1
0.46	0.0936	-2	0.0845	0	0.0793	-1
1.5	0.0899	3	0.0829	1	0.0788	0
4.7	0.0825	11	0.0729	13	0.0729*	7
15	0.0843	9	0.0631	25	0.057*	28
^1Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly. ^2Percent inhibition was calculated relative to the negative control mean (n=6). *Treatment group mean significantly different (Dunnett’s test, p<0.05) from the negative control mean. Only evaluated at 72 hours, per study protocol. Below are the conditions for the validity of the test: 1.      The mean cell density in the negative control flasks increased by a factor of 291 after three days, exceeding the 16x growth criterion. 2.      The coefficient of variation of average specific growth rate in the control replicates during the whole test period did not exceed 7%. It was 1.10%. 3.      The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) did not exceed 35%. It was 16.2%.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50, EyC50 and ErC50 values and their corresponding 95% confidence intervals were 7.1 (6.3 - 8.0), 7.1 (6.3 -8.0) and >15 (n/a*) mg a.i./L, respectively. The 72 -hour NOEC value, based on effects on cell density, yield and growth rate, was 1.5 mg a.i./L.
*95% confidence limits could not be calculated with the data obtained.

Executive summary:

The objective of this study was to determine the toxicity of syringonitrile to the freshwater green alga,Pseudokirchneriella subcapitata,during a 72-hour exposure period. The study protocol, "Syringonitrile: A 72-Hour Toxicity Test with the Freshwater Alga (Pseudokirchneriella subcapitata) was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 201:Freshwater Alga and Cyanobacteria, Growth Inhibition Test; Official Journal of the European Communities No. L383, Method C.3.Algal Inhibition Test,and the U.S. Environmental Protection Agency Series 850 -- Ecological Effects Test Guidelines, OPPTS Number 850.5400:Algal Toxicity, Tiers I and II. P. subcapitatawas exposed to six test concentrations and a negative control (culture medium) under static conditions for 72 hours. Six replicate test chambers in the control and three replicate test chambers in each treatment group were maintained. Nominal test concentrations were selected in based upon the results of an exploratory range finding toxicity test: 0.045, 0.14, 0.46, 1.5, 4.7, and 15 mg a.i./L. Measured concentrations were determined from samples of test medium collected from each treatment and control group at test initiation and at test termination (72 hours). At test initiation, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 P. subcapitata cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate, growth rates and yields. Cell densities and growth rates were used to calculate percent inhibition values relative to the negative control over the study period. EC50 and ErC50 values (i.e., the theoretical toxicant concentrations that would produce a 50% reduction in cell density and growth rate, respectively) were calculated. In addition, the EyC50 value (i.e., the theoretical toxicant concentration that would produce a 50% reduction in yield) was calculated for the 72-hour interval of the exposure period. No-observed-effect concentrations (NOEC) were determined at 72 hours through statistical evaluation of the cell density, yield, and growth rates, as well as examination of the concentration-response pattern.

After 72 hours of exposure, there were no signs or noticeable aggregation, flocculation, or adherence to the test chambers in the negative control or in any treatment groups. P. subcapitata cells in the treatment levels appeared normal when compared to cells present in the negative control.

In conclusion, the 72-hour EC50, EyC50 and ErC50 values and their corresponding 95% confidence intervals were 7.1 (6.3 - 8.0), 7.1 (6.3 -8.0) and >15 (n/a) mg a.i./L, respectively. The 72-hour NOEC value, based on effects on cell density, yield and growth rate, was 1.5 mg a.i./L.

Description of key information

Toxicity of 4-hydroxy-3,5-dimethoxybenzonitrile (syringonitrile) alone and a blend of 4-hydroxy-3,5-dimethoxybenzonitrile(syringonitrile)/laccase enzyme granules/adipic acid/monosodium phosphate (ready to use commercial product) to aquatic algae was examined per OECD 201.

Per ECHA's request under the decision number CCH-D-0000001424-81-05/F, "Growth inhibition study aquatic plants (algae) test (EU test method C3)" has been conducted. The key values entered below were obtained from this new study; discussion follows below.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.1 mg/L

EC10 or NOEC for freshwater algae:

1.5 mg/L

Additional information

Discussion re: newly obtained data (June 2012):

The form in the supply chain for syringonitrile is in conjunction with laccase enzyme, adipic acid, and monosodium phosphate, a ready-to-use product in the textile industry for denim bleaching applications (Tradename: PrimaGreen EcoFade LT100). This composition is referred to here as RTU blend. In order to examine the toxicity to aquatic algae of the 4 -hydroxy-3,5-dimethoxybenzonitrile (syringonitrile, subject of this registration) as well as the ready-to-use commercial product, two separate studies were conducted in accordance with OECD 201 guideline, Official Journal of the European Communities No. L383, Method C3, and the U.S.EPA Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.5400: Algal Toxicity, Tiers I and II.

Syringonitrile was tested on the growth of the freshwater algae, P. subcapitata in a 72 -hour test. EC50 was determined to be 7.1 mg a.i./L and a NOEC was determined to be 1.5 mg a.i./L. Under the GHS guideline, this is classified as H411 - chronic aquatic hazard category 2 (toxic to aquatic life with long lasting effects).

For the RTU blend, the exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 110 mg/L. The No Observed Effect Concentration based on biomass integral was 12.5 mg/L. Under GHS guideline, this classified the product as "not acutely toxic to algae." Please note that the new study was only conducted on the syringonitrile only and the RTU study referred here is the same study as what was originally submitted.

The RTU commercial blend is the only form of syringonitrile that will be commercialized by Genencor International for use in textiles bleaching. Exposure of the syringonitrile by itself to the aquatic environment is practically non-existent. After use, the mixture (from the RTU

blend) is released into the environment through the sewage system and any impact to the environment would result from exposure to this discharged mixture and not from syringonitrile by itself.

Using a weight-of-evidence approach, it can be concluded that any reaction products from syringonitrile in the discharge water after textiles application will not pose a threat to the aquatic algae.

 

Discussion from the original submission:

The form in the supply chain for 4-hydroxy-3,5-dimethoxybenzonitrile is in conjunction with laccase enzyme, adipic acid, and monosodium phosphate, a ready-to-use product in the textile industry for denim bleaching applications (Tradename: PrimaGreen EcoFade LT100). This composition is referred to here as RTU blend. In order to examine the toxicity to aquatic algae of the 4 -hydroxy-3,5 -dimethoxybenzonitrile (subject of this registration) as well as the ready-to-use commercial product, two separate studies were conducted in accordance with OECD 201 guideline. 4 -hydroxy-3,5 -dimethoxybenzonitrile was tested on the growth of the freshwater algae Selenastrum capricornutum in a 96-hour test. IC50 was determined to be 3.99 mg/L and a NOEC was determined to be <1 mg/L. Under GHS guideline, this is classified as category acute 1, very toxic to aquatic life.

For the RTU blend, the exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 110 mg/L. The No Observed Effect Concentration based on biomass integral was 12.5 mg/L. Under GHS guideline, this classified the product as "not acutely toxic to algae."

The RTU commercial blend is the only form of 4 -hydroxy-3,5 -dimethoxybenzonitrile that will be commercialized by Genencor International for use in textiles bleaching. Exposure of the 4 -hydroxy-3,5-dimethoxybenzonitrile by itself to the aquatic environment is practically non-existent.

After use, the mixture (from the RTU blend) is released into the environment through the sewage system and any impact to the environment would result from exposure to this discharged mixture and not to 4 -hydroxy-3,5–dimethoxybenzonitrile by itself.

Using a weight-of-evidence approach, it can be concluded that any reaction products from 4 -hydroxy-3,5 -dimethoxybenzonitrile in the discharge water after textiles application will not pose a threat to the aquatic algae.