Phenol, 4,4'-(1-methylethylidene)bis-, polymer with (chloromethyl)oxirane, reaction products with diethylenetriamine and 4-methyl-2-pentanone in xylene and butanol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PX708164
- Expiration date of the lot/batch: 30 June 2018 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accomodated Fraction: Preparation of test solutions started with loading rates individually prepared ranging between 0.10 and 100 mg/L. The test item was weighed on watch glasses which were added to the medium. A 7 - 8 hour period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure except for the highest test concentration of the range-finding test which was slightly hazy. After preparation, volumes of 40 mL were added to each replicate of the respective loading rate. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
- Eluate: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Differential loading: WAFs prepared at loading rates of 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Method of cultivation: 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.

ACCLIMATION
- Acclimation period: 3- 4 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.
- Culturing media and conditions (same as test or not): Adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

Between 22 and 23°C

pH:

7.6 ± 0.2

Nominal and measured concentrations:

WAFs prepared at loading rates of 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL
- Type : closed airtight
- Material, size, headspace, fill volume: all-glass, 40 mL
- Aeration: no
- Initial cells density: An initial cell density of 1 x 104 cells/mL
- Control end cells density: 165 x 104 cells/mL
- No. of vessels per concentration (replicates):3 replicates of each loading rate,1 replicate of each loading rate without algae.
- No. of vessels per control (replicates): 6 replicates of the control, 1 replicate without algae.

GROWTH MEDIUM
- Standard medium used: yes , adjusted M2

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: culkture M1, acclimation and test M2
- Intervals of water quality measurement: Beginning and end of test

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 79 to 83 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] 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 cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Chlorophyll measurement: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: 1.0 to 100 mg/L, increasing by a factor of 10 and to a control
- Test concentrations: 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L
- Results used to determine the conditions for the definitive study: Biologically relevant growth rate inhibition (i.e. >10%) was recorded at WAFs prepared at loading rates of 10 and 100 mg/L and biologically relevant yield inhibition was recorded at WAFs prepared at all loading rates. Respectively 75% and 99% growth rate and yield inhibition was recorded at the highest loading rate at the end of the test.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.071 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.6 mg/L

Individual cell densities (x10⁴cells/mL)

Time	Replicate	Setalux 7002 BX-55; WAF loading rate (mg/L)
		Control	0.10	0.32	1.0	3.2	10	32
0 h	1	1.000	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
n		6	3	3	3	3	3	3
Mean		1.0	1.0	1.0	1.0	1.0	1.0	1.0
St dev		0	0	0	0	0	0	0
CV		0	0	0	0	0	0	0
24 h	1	5.583	4.521	1.000	1.203	2.868	1.973	3.448
	2	9.009	6.554	1.000	1.724	7.578	2.911	4.656
	3	8.023	10.793	1.000	6.445	8.239	5.160	6.424
	4	6.266
	5	4.358
	6	4.781
n		6	3	3	3	3	3	3
Mean		6.3	7.3	1.0	3.1	6.2	3.3	4.8
St dev		1.8	3.2	0.0	2.9	2.9	1.6	1.5
CV		29.0	43.9	0.0	92.4	47.0	48.9	30.9
48 h	1	27.190	25.098	7.101	1.979	1.328	2.000	3.198
	2	26.719	25.699	6.776	4.342	1.599	2.456	3.572
	3	29.922	29.293	8.473	3.681	1.290	2.900	2.022
	4	28.697
	5	28.198
	6	27.505
n		6	3	3	3	3	3	3
Mean		28.0	26.7	7.5	3.3	1.4	2.5	2.9
St dev		1.2	2.3	0.9	1.2	0.2	0.5	0.8
CV		4.1	8.5	12.1	36.6	12.0	18.4	27.6
72 h	1	153.844	161.623	66.691	16.522	4.477	6.196	6.651
	2	163.531	158.186	69.347	25.233	5.101	6.716	6.825
	3	176.416	179.658	71.130	25.179	4.358	6.911	3.979
	4	168.648
	5	165.981
	6	160.479
n		6	3	3	3	3	3	3
Mean		164.8	166.5	69.1	22.3	4.6	6.6	5.8
St dev		7.6	11.5	2.2	5.0	0.4	0.4	1.6
CV		4.6	6.9	3.2	22.5	8.6	5.6	27.4

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 165. The test fulfils this validity criterion.

Individual Growth Rates (day-1)

Time	Replicate	Setalux 7002 BX-55; WAF loading rate (mg/L)
		Control	0.10	0.32	1.0	3.2	10	32
0-24 h	1	1.72	1.509	0	0.185	1.054	0.68	1.238
	2	2.198	1.88	0	0.545	2.025	1.068	1.538
	3	2.082	2.379	0	1.863	2.109	1.641	1.86
	4	1.835
	5	1.472
	6	1.565
n		6	3	3	3	3	3	3
Mean		1.812	1.923	0	0.864	1.729	1.13	1.545
St dev		0.2857	0.4366	0	0.8837	0.5866	0.4836	0.3112
CV		15.766	22.711		102.251	33.923	42.809	20.137
0-48 h	1	1.651	1.611	0.98	0.341	0.142	0.347	0.581
	2	1.643	1.623	0.957	0.734	0.235	0.449	0.637
	3	1.699	1.689	1.068	0.652	0.127	0.532	0.352
	4	1.678
	5	1.67
	6	1.657
n		6	3	3	3	3	3	3
Mean		1.666	1.641	1.002	0.576	0.168	0.443	0.523
St dev		0.0205	0.0416	0.0589	0.2071	0.0583	0.0931	0.1509
CV		1.232	2.536	5.883	35.982	34.685	21.02	28.829
0-72 h	1	1.679	1.695	1.4	0.935	0.5	0.608	0.632
	2	1.699	1.688	1.413	1.076	0.543	0.635	0.64
	3	1.724	1.73	1.422	1.075	0.491	0.644	0.46
	4	1.709
	5	1.704
	6	1.693
n		6	3	3	3	3	3	3
Mean		1.701	1.704	1.412	1.029	0.511	0.629	0.577
St dev		0.0154	0.0227	0.0108	0.0813	0.0281	0.0189	0.1014
CV		0.907	1.332	0.767	7.902	5.491	3.001	17.57

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.91%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test fulfils this validity criterion.

Validity criteria fulfilled:

yes

Conclusions:

The EL50 for growth rate inhibition (72h-ERL50) was 2.9 mg/L with a 95% confidence interval ranging from 2.7 to 3.2 mg/L.

Executive summary:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Setalux 7002 BX-55 reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAFs prepared at loading rates of 0.32 mg/L and higher.

The EL50 for growth rate inhibition (72h-ERL50) was 2.9 mg/L with a 95% confidence interval ranging from 2.7 to 3.2 mg/L.

The EL50 for yield inhibition (72h-EYL50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L.

The EL10 for growth rate inhibition (72h-ERL50) was 0.071 mg/L with a 95% confidence interval ranging from 0.056 to 0.088 mg/L.

The EL10 for yield inhibition (72h-EYL50) was 0.11 mg/L with a 95% confidence interval ranging from 0.10 to 0.12 mg/L.

The 72h-NOELR for growth rate and yield inhibition was 0.10 mg/L based on statistical significance and biological relevance.

Description of key information

The objective of the study was to evaluate Setalux 7002 BX-55 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10 and EL50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Setalux 7002 BX-55 tested was a clear dark yellow viscous liquid mixture of different components and not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared a. A final test was performed based on a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 0.10, 0.32, 1.0, 3.2, 10 and 32 mg Setalux 7002 BX-55 per litre. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours.

Growth rates and yield were in the range of the controls at the lowest WAF during the 72 -hour test period, whereas inhibition of growth rate and yield at the five highest WAFs were statistically significant. Overall, the biological results did not follow a straight dose-response curve at the three highest loading rates but rather approached an asymptote around 70% inhibition for growth rate with almost maximum inhibition for yield.

The project did not include sampling for determination of actual exposure concentrations because no analytical method could be developed for this complex UVCB. However, the biological results indicate algae were exposed to the test item. Consequently, results are reported on loading rates.

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

The effect parameters obtained in this study are:

The EL50 for growth rate inhibition (72h-ERL50) was 2.9 mg/L with a 95% confidence interval ranging from 2.7 to 3.2 mg/L.

The EL50 for yield inhibition (72h-EYL50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L.

The EL10 for growth rate inhibition (72h-ERL50) was 0.071 mg/L with a 95% confidence interval ranging from 0.056 to 0.088 mg/L.

The EL10 for yield inhibition (72h-EYL50) was 0.11 mg/L with a 95% confidence interval ranging from 0.10 to 0.12 mg/L.

The 72h-NOELR for growth rate and yield inhibition was 0.10 mg/L based on statistical significance and biological relevance.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.9 mg/L

EC10 or NOEC for freshwater algae:

0.071 mg/L