Rosin, reaction products with acrylic acid

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:

16-Feb-2018 to 08-Mar-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Rosin, reaction products with acrylic acid

EC Number:

280-192-2

EC Name:

Rosin, reaction products with acrylic acid

Cas Number:

83137-13-7

Molecular formula:

N.A. - UVCB substance

IUPAC Name:

Rosin, reaction products with acrylic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 646729
- Physical state: yellow to brownish solid
- Expiration date of the lot/batch: 19 November 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Sampling and analysis

Analytical monitoring:

yes

Remarks:

MTDID 28640 is a complex mixture (UVCB), therefore Total Organic Carbon (TOC) analysis was used as a proxy for the chemical.

Details on sampling:

- Concentrations: All; sampled at test initiation, and at test termination.
- Sampling method: Aliquots from test initiation were collected from the bulk test solutions. Aliquots from test completion were collected from pooled volumes of each of the control and test solution replicates.
- Sample storage conditions before analysis: Samples were collected in glass containers and stored refrigerated (2°C to 8°C) until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:Test solutions were prepared as water soluble fractions (WAFs) individually by adding the weighed amount of MTDID 28640 to approximately 900 mL of dilution water (test medium), then adding additional dilution water to bring the volume to 1.8 L. The control was prepared in a similar manner; however, no MTDID 28640 was added. Each solution was stirred using a stir plate and stir bar to create a vortex (~15%) and covered to reduce contaminants. Stirring occurred for approximately 92 hours. Solutions were then filtered through a 0.45 μm membrane filter in a glass vacuum filtration apparatus.
- Controls: Negative (Lab) Control: Blank test medium; Negative (Procedural) Control: Blank test medium prepared as WAF test solutions
- Evidence of undissolved material: At the time of preparation, test item WAFs appeared clear and colorless. In higher loading rate WAFs, visible, undissolved MTDID 28640 was observed spinning in the water column. After the 92 hour stirring period, there was no change in appearance. After filtration, all test solutions and controls appeared clear and colorless with no visible MTDID 28640. Test solutions and controls appeared clear and colorless at test inititation and remained consistently clear and colorless until approximately the 48-hour observation time point. At test completion, all test solutions appeared green due to algal growth.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CPCC 37
- Source: In-house culture; original culture was obtained from Canadian Phycological Culture Center (Waterloo, ON) on March 02, 2017
- Method of cultivation: The starter culture was used to initiate new algae cultures using nutrient medium (similar to AAP-medium; USEPA)
NaNO3, 25500 µg/l
MgCl2∙6H2O, 10000 µg/l
CaCl2∙2H2O, 4420 µg/l
H3BO3, 186 µg/l
MnCl2∙4H2O, 416 µg/l
ZnCl2, 3.28 µg/l
CoCl2∙6H2O, 1.43 µg/l
CuCl2∙2H2O, 0.012 µg/l
Na2MoO4∙2H2O, 7.26 µg/l
FeCl3∙6H2O, 160 µg/l
Na2EDTA∙2H2O, 300 µg/l
MgSO4∙7H2O, 14700 µg/l
K2HPO4, 1044 µg/L
NaHCO3, 15500 µg/l

ACCLIMATION
Algal culture medium was the same as the test medium. Culture used to inoculate test vessels was in exponential growth phase and contained approximately 0.98E+06 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 °C

pH:

7.6 to 8.0

Conductivity:

90 μS/cm to 94 μS/cm

Nominal and measured concentrations:

Nominal; 0 mg/L (Negative control), 0 mg/L (WAF control), 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L
Measured; see Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL, all-glass, covered with foam stopper. Fill volume was 50 mL
- Agitation: Yes, during incubation the algal cells were mixed on a horizontal shaker at approximately 75 rpm.
- Initial cells density: 0.98E+04 cells/mL
- Control end cells density: 101.7E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (plus one extra vessel at each loading rate for possible TOC analysis at 72 hours)
- No. of vessels per control (replicates): 6 (plus one extra vessel for possible TOC analysis at 72 hours)
GROWTH MEDIUM
- Standard medium used: Similar to AAP-medium (USEPA)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Millipore deionised water system (Millipore RiOs16; Millipore Milli-Q Academic)
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH at test initiation and completion, conductivity at test initiation, temperature of test chamber monitored and recorded daily
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous illumination with light intensity within the range of 5642 to 6006 lux.
EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells were counted with a Cellometer X4HM Auto Counter set to count fluorescent algae-PKS cell type, with dilution factor of 1. The cell density in each replicate was determined by counting a minimum of four aliquots from each test vessel. An unusual unusual appearance of the algal cells, including observed differences in cell size, was noted, if present.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations:0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

zinc sulphate heptahydrate (CAS# 7446-20-0)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes. The CV of the mean specific growth rates during the whole test period in the control was 2.76%. For the specific growth rates of each time period (days 0-1, 1-2. and 2-3) the highest mean CV in the control was 23.36%.
- Observation of abnormalities (for algal test): none reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not applicable
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.079 mg/L (95% Confidence limits, 0.063 mg/L to 0.098 mg/L) (cell yield). Historical range (± 2 Std. Dev.) for the reference substance at the contract lab lies between 0.033 mg/L and 0.111 mg/L.
- Other: Reference substance toxicity assay conducted seven months prior to the MTDID 28640 assay.

Reported statistics and error estimates:

Growth rate data were normally distributed by Shapiro-Wilk's test, Shapiro-Wilk´s W = 0.9691, p(W) = 0.7128 (>0.01). Variance of growth rate data were equal by Bartlett's Equality of Variance Test, Test statistic = 3.464, P-value = 0.6289. Statistical calculations were done using CETIS statistical software v.1.9.2.4. (Tidepool Scientific Software, McKinleyville, CA, USA).

Any other information on results incl. tables

Table 2, Mean Cell Counts in Each 24-hour Period

Nominal Loading Rate	Mean Cell Counts (x10E+04 cells/mL)
(mg/L)	0 -h	24 -h	48 -h	72 -h
Negative Control (0)	0.98	3.9	19.4	101.7
WSF Control (0)	0.98	4.2	19.7	130.3
6.25	0.98	4.2	21.7	140.5
12.5	0.98	5.1	21.1	150.3
25	0.98	3.9	23.4	152.3
50	0.98	3.5	23.3	147.3
100	0.98	2.7	20.8	110.5

Table 3, Summary of the Mean Inhibition (%) in Cell Yield and Specific Growth Rate

Nominal Loading Rate of MTDID 28640 (mg/L)	Mean Specific Growth Rate (1/day)	Mean Inhibition in Specific Growth Rate (%) v. negative control
Negative Control (0)	1.55	not applicable
WSF Control (0)	1.63	-5.18
6.25	1.65	-6.90
12.5	1.67	-8.07
25	1.68	-8.68
50	1.67	-8.09
100	1.58	-1.89

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by a factor of > 16 (104); The mean CV for section-by-section specific growth rate in the controls was < 35% (23.36%); the CV of average specific growth rates in the whole test period for controls was < 7% (2.76%).

Conclusions:

The 72-hour EC50 & EC10 (growth rate) of MTDID 28640 to Pseudokirchneriella subcapitata was beyond the range tested (water soluble fraction prepared at 100 mg/L (nominal) loading level). The test was conducted per OECD TG 201.

Executive summary:

The toxicity of MTDID 28640 to the freshwater algae Pseudokirchneriella subcapitata was assessed in a limit test according to OECD201. MTDID 28640 is a UVCB substance with low water solubility, therefore water soluble fractions with loading rates of 6.25, 12.5, 25, 50, and 100 mg/L were prepared. Analytical measurement of total organic carbon (TOC) concentration as a proxy for MTDID 28640 was conducted, to confirm presence of dissolved substance over the period of the test. The 72-hour EC10 and EC50 were > 100 mg/L (nominal concentration), and therefore greater than the water solubility of MTDID 28640 in the test medium. The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria. The test is considered reliable without restrictions, and the results are suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.