Reaction mass of 2,2,4-trimethylhexane-1,6-diol and 2,4,4-trimethylhexane-1,6-diol

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

Study period:

2016-02-23 to 2016-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25 - 12.50 - 25.0 - 50.0 - 100 mg/L
- Sampling method: The concentrations of the test item were analytically verified from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Stock solution: A stock solution of 100 mg/L was freshly prepared with dilution water.

Media preparation: direct addition of the test item and shaking until the test item was completely dissolved.

Test concentrations: 5 concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.
- Culture medium: nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

0.24 mmol Ca+Mg/l

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x E03 - E04 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 1592351 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 75.3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 12 45
10.0 -1 -5
1.00 -8 -56

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.7 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
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: 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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50-Values of the Reference Item based on nominal concentrations [mg/L], (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.564 0.797 ± 0.428
95 % confidence interval 0.546 - 0.582

Yield inhibition
EyC50 0.322 0.415 ± 0.275
95 % confidence interval 0.239 - 0.342

SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. Detection of statistically significant differences was considered negligible when inhibition values were ≤ 10 %. These findings were classified as “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals
EC10-, EC20- and EC50-values were carried out using standard procedures.

Cell densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6305	5242	5620	4526
	2	6305	5682	5902	6412
	3	6305	5327	6302	4574
	Mean	6305	5417	5941	5171
50.0	1	6305	10438	23955	76118
	2	6305	11393	25181	70370
	3	6305	10942	22875	43975
	Mean	6305	10924	24004	63488
25.0	1	6305	14260	76718	715064
	2	6305	17215	127094	1084155
	3	6305	15565	82689	838113
	Mean	6305	15680	95500	879111
12.5	1	6305	16896	142925	1092415
	2	6305	18937	171369	1530995
	3	6305	18263	134434	1160071
	Mean	6305	18032	149576	1261160
6.25	1	6305	17747	152092	1176703
	2	6305	22655	204520	1560651
	3	6305	26946	234571	1637356
	Mean	6305	22449	197061	1458237
Control	1	6305	28186	229091	1505934
	2	6305	27639	245740	1751356
	3	6305	24217	205761	1567670
	4	6305	22892	164440	1353990
	5	6305	29145	248587	1594338
	6	6305	30777	224022	1780815
	Mean	6305	27143	219607	1592351

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		-0.111	100		-1779	100
	2		0.006	100		107	100
	3		-0.107	100		-1731	100
	Mean	(+)	-0.071	100	(+)	-1134	100
50.0	1		0.830	55		69813	96
	2		0.804	56		64065	96
	3		0.647	65		37670	98
	Mean	(+)	0.761	59	(+)	57183	96
25.0	1		1.58	14		708759	55
	2		1.72	7		1077850	32
	3		1.63	12		831808	48
	Mean	(+)	1.64	11	(+)	872806	45
12.5	1		1.72	7		1086110	32
	2		1.83	1		1524690	4
	3		1.74	6		1153766	27
	Mean	(-)	1.76	4	(-)	1254855	21
6.25	1		1.74	5		1170398	26
	2		1.84	0		1554346	2
	3		1.85	-1		1631051	-3
	Mean	(-)	1.81	2	(-)	1451932	8
Control	1		1.83			1499629
	2		1.88			1745051
	3		1.84			1561365
	4		1.79			1347685
	5		1.84			1588033
	6		1.88			1774510
	Mean		1.84			1586046

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.50	2.10	1.88	1.83	0.303	16.6	19.0
	2	1.48	2.19	1.96	1.88	0.362	19.3
	3	1.35	2.14	2.03	1.84	0.430	23.4
	4	1.29	1.97	2.11	1.79	0.439	24.5
	5	1.53	2.14	1.86	1.84	0.307	16.6
	6	1.59	1.99	2.07	1.88	0.260	13.8
				Mean	1.84
				SD ±	0.03
				CV [%]	1.83

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L. All effect level are based on nominal concentration which had been analytically verified.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3.The aim of the study was the determination of NOEC, LOEC, EC₁₀-, EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 100 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 2.0 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

The concentrations of the test subsance were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations in the fresh media (0 h) were in the range of 111 to 118% of the nominal values at tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 106 to 120% of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations. In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L.

Description of key information

The toxicity of the test substanceto the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009/Method C.3.The aim of the study was the determination of NOEC, LOEC, EC₁₀-, EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 100 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 2.0 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

The concentrations of the test subsance were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations in the fresh media (0 h) were in the range of 111 to 118% of the nominal values at tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 106 to 120% of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations.In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

45.7 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L