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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2011-04-11 to 2011-04-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of concentrations the lowest, middle and the highest test concentration and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates of the lowest, middle and highest test concentration were prepared without algae for analysis after 0 and 72 hours. Additionally two replicates of the middle concentration with algae (incubated under test conditions) were analysed after 72 hours. Sorption to the walls of the glass container was checked too.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with dilution water
- Eluate: Dilution water acc. to the guideline
- Differential loading: 0.015 - 0.034 - 0.075 - 0.165 - 0.364 - 0.800 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: 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): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 hours algae were transferred from the nominal concentrations 0.0750 – 0.800 mg/L and the control into fresh untreated medium. 0.2 – 0.5 mL algae suspension from each test replicate and from two control replicates were filled up with dilution water to 10 mL and allowed to grow for further 4 – 7 days to determine whether the effect of the test item was reversible. For concentration levels 0.364 and 0.800 mg/L 45 mL algae suspension from each replicate were centrifuged (3000 rpm, 15 min). Aliquots were taken from the sedimented phase.

Hardness:

Not measured

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
0.800 8.34 8.29
0.364 8.33 8.31
0.165 8.33 8.31
0.0750 8.28 8.28
0.0340 8.30 8.28
0.0150 8.26 8.27
Control 8.26 8.29

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 9846 cells/mL
- Control end cells density: Mean 1929863 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline

Table : Composition of Dilution Water

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-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


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



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 hours)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10 100 100
1 100 100
0.1 25 73
0.01 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.641 (CI 0.618 - 0.664) mg/L
Yield Inhibition: 0.352 (CI 0.336 - 0.367) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by maximum likelihood regression using the probit transform.
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. DUNNETT’s multiple comparison test was used. The SHAPIRO-WILK-Test was used to test for normally distributed populations. Variances were tested by the BARTLETT’S test.

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of DDAC (0-72 h)

based on nominal test item concentrations [mg/L]  

	Rate-related inhibition
NOEC	0.0340
LOEC	0.0750
ErC10	0.0514 (0.00848 – 0.0862)
ErC20	0.0753 (0.0214 – 0.114)
ErC50	0.156 (0.0981 – 0.249)
	Inhibition of yield
NOEC	0.0150
LOEC	0.0340
EyC10	0.0128 (0.00137 – 0.0223)
EyC20	0.0195 (0.00408 – 0.0311)
EyC50	0.0435 (0.0252 – 0.0723)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
0.800		1		9846		9846	9846	9846
		2		9846		9846	9846	9846
		3		9846		9846	9846	9846
		Mean		9846		9846	9846	9846
0.364		1		9846		9846	9846	9846
		2		9846		9846	9846	9846
		3		9846		9846	9846	30888
		Mean		9846		9846	9846	16860
0.165		1		9846		11284	54242	399927
		2		9846		9846	23495	169580
		3		9846		9846	21352	164880
		Mean		9846		9846	33030	244796
0.0750		1		9846		14104	60865	470192
		2		9846		20416	72112	492235
		3		9846		13465	69249	485467
		Mean		9846		15995	67409	482631
0.0340		1		9846		23702	142104	1051535
		2		9846		26672	180602	1214108
		3		9846		30564	216594	1360184
		Mean		9846		26979	179767	1208609
0.0150		1		9846		31884	234351	1367140
		2		9846		37181	299526	1922351
		3		9846		33012	260723	1695576
		Mean		9846		34026	264867	1661689
Control		1		9846		38196	309504	2137893
		2		9846		39503	344815	1981712
		3		9846		39320	330226	2093384
		4		9846		38723	262631	1754185
		5		9846		37548	261512	1655579
		6		9846		40509	295822	1956426
		Mean		9846		38967	300752	1929863

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	Repli-cate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.800	1		0.000	100		0	100
	2		0.000	100		0	100
	3		0.000	100		0	100
	Mean	(+)	0.000	100	(+)	0	100
0.364	1		0.000	100		0	100
	2		0.000	100		0	100
	3		0.381	78.3		21042	98.9
	Mean	(+)	0.127	92.8	(+)	7014	99.6
0.165	1		1.24	29.8		390081	79.7
	2		0.949	46.0		159734	91.7
	3		0.939	46.6		155034	91.9
	Mean	(+)	1.04	40.8	(+)	234950	87.8
0.0750	1		1.29	26.7		460346	76.0
	2		1.30	25.8		482389	74.9
	3		1.30	26.1		475621	75.2
	Mean	(+)	1.30	26.2	(+)	472785	75.4
0.0340	1		1.56	11.4		1041689	45.8
	2		1.61	8.71		1204262	37.3
	3		1.64	6.55		1350338	29.7
	Mean	(+)	1.60	8.90	(+)	1198763	37.6
0.0150	1		1.64	6.46		1357294	29.3
	2		1.76	0.00		1912505	0.39
	3		1.72	2.38		1685730	12.2
	Mean	(-)	1.71	2.95	(-)	1651843	14.0
Control	1		1.79			2128047
	2		1.77			1971866
	3		1.79			2083538
	4		1.73			1744339
	5		1.71			1645733
	6		1.76			1946580
	Mean		1.76			1920017

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	1.36	2.09	1.93	1.79	0.388	21.6	19.8
	2	1.39	2.17	1.75	1.77	0.389	22.0
	3	1.39	2.13	1.85	1.79	0.375	21.0
	4	1.37	1.91	1.90	1.73	0.310	18.0
	5	1.34	1.94	1.85	1.71	0.324	19.0
	6	1.41	1.99	1.89	1.76	0.307	17.4
				Mean	1.76
				SD ±	0.03
				CV [%]	1.91

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 72 h ErC50 and ErC10 values of C12-14 TMAC for Pseudokirchneriella subcapitata can be considered to be equivalent to 0.062 and 0.02 mg a.i./L respectively (nominal).

Executive summary:

A study was conducted to determine the acute toxicity study of the read across substance, DDAC (39.6% active ingredient), to freshwater green alga, Pseudokirchneriella subcapitata, according to OECD Guideline 201, in compliance with GLP. Algae (with initial cell density of 1 x 104cells/mL) were exposed to the test substance at nominal concentrations of 0.0150, 0.0340, 0.0750, 0.165, 0.364 and 0.800 mg/L for 72 h under static conditions and effects on growth rate and yield were determined. Three replicates were used to determine alga growth inhibition whereas six replicates were used for the control without addition of test substance. Analytical determinations of the test concentrations were performed. The measured concentrations at test start and end were in the range of 82-93% and 81-84% of the nominal values, respectively, indicative of stability of the test substance. Adsorption to glass was additionally checked for the test concentration of 0.165 mg/L, which was observed to be 16% of the applied amount after 72 h. Therefore it can be concluded that the algae were exposed to ≥ 80% of the nominal concentration of the test substance during the test. All effect values are given based on nominal test substance concentrations. The test substance was found to inhibit the growth and yield of the freshwater green algae after 72 h with the following effect values: The 72 h ErC50 and ErC10 values with 95% confidence intervals for inhibition of specific growth rate were 0.156 (0.0981 – 0.249) mg/L (i.e., equivalent to 0.062 mg a.i./L) and 0.0514 (0.00848 – 0.0862) mg/L (i.e. 0.020 mg a.i./L) respectively. The 72 h EyC50 and EyC10 values with 95% confidence intervals for inhibition of yield were 0.0435 (0.0252 – 0.0723) (i.e., equivalent to 0.0174 mg a.i./L) and 0.0128 (0.00137 – 0.0223) mg/L (i.e. 0.0051 mg a.i./L) respectively. The respective NOEC values were established at 0.034 mg/L (i.e. 0.013 mg a.i./L) and 0.0150 (i.e. 0.06 mg a.i./L) respectively. The results of the study can be considered as valid and the validity criteria were fulfilled. Under the conditions of the study, the 72 h ErC10 and ErC50 values for inhibition of specific growth rate of freshwater algae were determined to be at 0.02 and 0.062 mg a.i./L, respectively. The corresponding NOEC was established at 0.013 mg a.i./L (Scheerbaum, 2011). Based on the results of the read across study, the 72 h ErC50 and ErC10 values of C12-14 TMAC for Pseudokirchneriella subcapitata can be considered to be equivalent to 0.062 and 0.02 mg a.i./L respectively (nominal).

Description of key information

Based on the results of the read across study, the 72 h ErC50 and ErC10 values of 0.062 and 0.02 mg a.i./L respectively (nominal) for Pseudokirchneriella subcapitata has been considered further for hazard assessment for C12-14 TMAC.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.062 mg/L

EC10 or NOEC for freshwater algae:

0.02 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the read across substance, DDAC (39.6% active ingredient), to freshwater green alga, Pseudokirchneriella subcapitata, according to OECD Guideline 201, in compliance with GLP. Algae (with initial cell density of 1 x 104cells/mL) were exposed to the test substance at nominal concentrations of 0.0150, 0.0340, 0.0750, 0.165, 0.364 and 0.800 mg/L for 72 h under static conditions and effects on growth rate and yield were determined. Three replicates were used to determine alga growth inhibition whereas six replicates were used for the control without addition of test substance. Analytical determinations of the test concentrations were performed. The measured concentrations at test start and end were in the range of 82-93% and 81-84% of the nominal values, respectively, indicative of stability of the test substance. Adsorption to glass was additionally checked for the test concentration of 0.165 mg/L, which was observed to be 16% of the applied amount after 72 h. Therefore it can be concluded that the algae were exposed to ≥ 80% of the nominal concentration of the test substance during the test. All effect values are given based on nominal test substance concentrations. The test substance was found to inhibit the growth and yield of the freshwater green algae after 72 h with the following effect values: The 72 h ErC50 and ErC10 values with 95% confidence intervals for inhibition of specific growth rate were 0.156 (0.0981 – 0.249) mg/L (i.e., equivalent to 0.062 mg a.i./L) and 0.0514 (0.00848 – 0.0862) mg/L (i.e. 0.020 mg a.i./L) respectively. The 72 h EyC50 and EyC10 values with 95% confidence intervals for inhibition of yield were 0.0435 (0.0252 – 0.0723) (i.e., equivalent to 0.0174 mg a.i./L) and 0.0128 (0.00137 – 0.0223) mg/L (i.e. 0.0051 mg a.i./L) respectively. The respective NOEC values were established at 0.034 mg/L (i.e. 0.014 mg a.i./L) and 0.0150 (i.e. 0.06 mg a.i./L) respectively. The results of the study can be considered as valid and the validity criteria were fulfilled. Under the conditions of the study, the 72 h ErC10 and ErC50 values for inhibition of specific growth rate of freshwater algae were determined to be at 0.02 and 0.062 mg a.i./L, respectively. The corresponding NOEC was established at 0.014 mg a.i./L (Scheerbaum, 2011). Based on the results of the read across study, the 72 h ErC50 and ErC10 values of C12-14 TMAC for Pseudokirchneriella subcapitata can be considered to be equivalent to 0.062 and 0.02 mg a.i./L respectively (nominal).