Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

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:

02 June 2017 - 20 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

Molecular formula:

not applicable

IUPAC Name:

Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: Barium salts of resin acids and rosin acids
Appearance: light tan powder
Storage conditions: at room temperature desiccated

Specific details on test material used for the study:

Stability in water: stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) 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 checked by running a test vessel at the limit 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Direct application to the test medium: preparation of test solutions started with loading rates individually prepared at a range of 1.0 to 100 mg/L. A 3-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Subsequently, the aqueous Water Accommodated Fractions (WAFs) were collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

CULTIVATION:
- Method: 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/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

ACCLIMATION
- Acclimation period: 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Acclimation medium different from test medium: no, M2 (according to OECD 201)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 mg/L

Test temperature:

23 - 24°C

pH:

At test start: 7.6 - 8.1
At test end: 7.5 - 7.6

Nominal and measured concentrations:

Nominal test concentrations: WAFs prepared at loading rates of 10, 18, 32, 32 (without algae), 56 and 100 mg/L
Measured test concentrations (TWA): 1.9, 3.6, 6.5, 8.1, 13 and 19 mg/L
Since the concentrations at the end of the test decreased to 0.020-0.043% of the initial concentrations measured at the start of the test, the Time Weighted Average (TWA) concentrations were calculated.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 294 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 or 2 extra replicates of each test concentration without algae

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 82 to 91 µE/m^2/s

EFFECT PARAMETERS MEASURED: growth rate at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. Temperature: continuously in a temperature control vessel; Appearance of the cells: at the end of the final test microscopic observations were performed on the WAF prepared at 56 mg/L and control to observe for any abnormal appearance of the algae.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, growth rate was not inhibited at the two lowest test concentrations, while inhibition was >25% in the highest concentration. The expected EC50 for growth rate inhibition was above the exposure concentration in a WAF prepared at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (June 2017)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 6.1-6.6 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no

- Statistical relevant effects at the two lowest dose levels were found. These effects were below 10% and considered not biologically relevant. Based on biological relevance the NOEC was set on a dose level of 3.6 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72h-ErC50: 0.99 mg/L (95% CI: 0.97-1.0 mg/L)
- Other: the result fell within the historical data range (0.82 - 2.3 mg/L).

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 Measured concentrations versus nominal concentrations

Barium salts of resin acids and rosin acids concentration (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
10	7.11	4.47	0.00221	1.9
18	11.4	9.64	0.00485	3.6
32	21.6	16.8	0.00422	6.5
32^a	24.1	22.6	0.00920	8.1
56	41.7	33.4	0.0114	13
100	58.1	52.2	0.0209	19

^awithout algae

Table 2 Individual growth rates per day

Time	Replicate	Barium salts of resin acids and rosin acids; TWA conc. (mg/L)
		Control	1.9	3.6	6.5	13	19
0-24 h	1	2.006	2.08	2.178	2.041	1.604	1.334
	2	1.968	2.104	2.054	2.094	1.705	1.359
	3	1.967	2.224	2.061	2.045	1.603	1.338
	4	2.012
	5	2.02
	6	1.961
Mean:		1.989	2.136	2.098	2.06	1.637	1.344
Std.Dev.:		0.0263	0.0772	0.0698	0.0295	0.059	0.0138
n:		6	3	3	3	3	3
CV:		1.3	3.6	3.3	1.4	3.6	1
0-48 h	1	2.003	1.955	1.945	1.91	1.717	1.297
	2	2.051	1.973	1.91	1.864	1.723	1.394
	3	2.007	1.981	1.896	1.982	1.745	1.461
	4	1.959
	5	2.036
	6	1.985
Mean:		2.007	1.97	1.917	1.919	1.728	1.384
Std.Dev.:		0.0334	0.0136	0.0254	0.0593	0.0149	0.0825
n:		6	3	3	3	3	3
CV:		1.7	0.7	1.3	3.1	0.9	6
0-72 h	1	1.892	1.803	1.779	1.697	1.391	1.001
	2	1.902	1.821	1.744	1.682	1.413	0.987
	3	1.917	1.827	1.77	1.723	1.429	1.024
	4	1.864
	5	1.91
	6	1.878
Mean:		1.894	1.817	1.764	1.701	1.411	1.004
Std.Dev.:		0.02	0.0124	0.0182	0.0205	0.0191	0.0184
n:		6	3	3	3	3	3
CV:		1.1	0.7	1	1.2	1.4	1.8

Table 3 Growth rate and percentage inhibition for the total test period

Barium salts of resin acids and rosin acids TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.894	0.0200	6
1.9	1.817	0.0124	3	4.0*^#
3.6	1.764	0.0182	3	6.8*^#
6.5	1.701	0.0205	3	10.2*
13	1.411	0.0191	3	25.5*
19	1.004	0.0184	3	47.0*

* effect was statistically significant; ^#effect biologically not relevant (<10%)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks' section.

Conclusions:

The 72h-EC50 for growth rate was beyond the range tested, i.e. exceeded a concentration of 19 mg/L and the 72h-NOEC for growth rate inhibition was 3.6 mg/L, both based on the TWA of measured concentrations.

Executive summary:

A study was performed to assess the effect of Barium salts of resin acids and rosin acids on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP.

 

A combined limit/range-finding study was performed with test solutions prepared individually at loading rates of 1.0, 10 and 100 mg/L by applying an 3 -day period of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters, whereafter the Water Accommodated Fractions (WAFs) were used for testing. The same preparation of test solutions was used for the final test.

 

In the final test, six replicates of exponentially growing algal cultures with an initial algal cell density of 10^4cells/mL, were exposed to a WAF prepared at loading rates of 1.9, 3.6, 6.5, 13 and 19 mg/L. A blank control and a replicate without algae were included. Samples were taken from all test concentrations and the blank control and analysed for confirmation of actual exposure concentrations at the start and after 24 and 72 hours of exposure. Test concentrations were 0.020 - 0.043% in the samples taken at t=72 h. To determined the effect concentrations, the Time Weighted Average (TWA) exposure concentrations were calculated to be 1.9, 3.6, 6.5, 8.1, 13 and 19 mg/L.

Cell densities were recorded to determine the inhibition of growth rate. The 72h-ErC50 of Barium salts of resin acids and rosin acids was >19 mg/L and the 72h-NOErC of Barium salts of resin acids and rosin acids was 3.6 mg/L.

All acceptability criteria were met and therefore the study was considered to be valid.