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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-05-21 to 2013-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2 ml
Storage Samples were stored in a freezer 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 demonstrated by running a test vessel at all concentrations but without algae and samples for analysis were taken at the start, after 24 hours of exposure (only from the highest concentration) and at the end of the test period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For the preparation of test solutions test medium containing 4 mg humic acid per litre (IHSS Suwannee River, USA) was preheated to approximately 60°C. Into this medium 25 mg of MDIPA-Esterquat C16-18 and C18 unsatd. (55°C - 60°C) was added in small amounts. After the dosage the stirring was continued for a further 5 to 10 minutes. Thereafter, the dispersion was left to cool down to room temperature. The lower test concentrations were prepared by subsequent dilutions of the 25 mg/l concentration in test medium. All test vessels were pre-incubated for 30 minutes with the respective test solution before the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata, NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture.
- Age of inoculum (at test initiation): 3-4 d (final test), 3 d (range-finding test)
- Method of cultivation: 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
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
main test: 21-24°C, constant within 2°C
Range-finding test: The temperature has been just below the optimum range of 21-24°C during the test period, i.e. temperature ranged between 20.1 and 21.4°C.
pH:
8.0-8.3
Nominal and measured concentrations:
nominal: 0, 0.25, 0.80, 2.5, 8.0 and 25 mg/L
(+ 4 mg humic acid per litre (<2 mg/L DOC))
measured (TWA): 0.29, 0.91, 2.7, 6.2 and 25 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1E04 cells/mL
- Control end cells density: 103.9E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 with the addition of 4 mg humic acid per litre

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: TLD-lamps type ‘Cool-white’ 30 Watt, light intensity 62 to 72 µE/m²/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Other: At the end of the test microscopic observations were performed on the lowest test concentration (range-finding test) or on the test concentration closest to the EC50 (both final tests) and 1 lower concentration (final test with humic acid) to observe for any abnormal appearance of the algae

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 (sqrt(10))
- Range finding study, concentrations nominal 0.025, 0.25, 2.5, 25 mg/L
- Test concentrations: main study: nominal 0.25, 0.80, 2.5, 8.0 and 25 mg/L, with humic acid
- Results used to determine the conditions for the definitive study: Percentage reduction of growth rate and inhibition of yield during the range-finding test for nominal 0.025, 0.25, 2.5, 25 mg/L
2.5mg/L: 4.6% reduction growth rate, 17.3% reduction yield
25mg/L: 100% reduction growth rate, 100% reduction yield
Based on these results samples taken from nominal 0.25 and 25 mg/L were analysed. The initial concentrations were 0.29 and 28 mg/L, respectively. The lower concentration remained stable during the test period (96% of initial at the end of the test). The higher concentration remained stable during the first 24 hours of exposure (91% of initial) but slightly decreased towards the end (78% of initial). The expected EC50 for both growth rate reduction and yield inhibition was between nominal concentrations of 2.5 and 25 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% ci 2.5-4.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% ci 6.4-10 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.91 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% ci 0.45-3.7 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% ci 1.6-12 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of test revealed no abnormalities

After 48 hours of exposure a brown layer was observed sticking to the glass just above the medium surface at an average concentration of 1.7 mg/L. After 72 hours of exposure a light floating layer and precipitate was observed at an average concentration of 0.39 mg/L; a floating layer, precipitate and a brown layer was observed sticking to the glass just above the medium surface at average exposure concentrations of 1.3 and 1.7 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50 = 0.97 mg/L, 95% confidence interval 0.62 to 1.5 mg/L; historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L

Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. 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 reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

At the start of the test, the actual test concentrations were slightly above nominal (122-136%). These concentrations remained stable during the first 24 hours of exposure (83-92% of initial, except for nominal 8.0 mg/l that decreased to 66% of initial. All concentrations decreased towards the end of the test (37-82% of initial. Given theseresults, the average exposure concentrations were calculated to be: 0.29, 0.91, 2.7, 6.2 and 25 mg/l. At the end of the test, concentrations without algae were generally higher than concentrations with algae indicating that part of the test substance adsorbs to the algal cells.

At the start of the test measured DOC concentrations ranged between 1.6 and 2.9 mg/l for all concentrations up to and including 2.7 mg/l. These concentrations remained stable during the test. Measured DOC at 6.2 mg/l was 6.4 mg/l at the start of the test and decreased to 1.2 mg/l at the end of the test. Measured DOC at 25 mg/l was 12 mg/l at both the start and the end of the test.

Mean cell densities (x 104cells/ml) during the final test

Average conc.

Exposure time (hours)

 

(mg/l)

0

24

48

72

control

1.0

5.5

21.7

103.9

0.29

1.0

5.4

22.5

99.3

0.91

1.0

5.9

22.8

104.1

2.7

1.0

4.2

17.6

83.8

6.2

1.0

1.3

6.3

20.5

25

1.0

1.0

1.0

1.0

Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Average conc.

Mean growth rate

Yield (0-72 h)

 

(mg/l)

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.06447

102.88

0.29

0.06383

1.0

98.33

4.4

0.91

0.06452

-0.1

103.08

-0.2

2.7

0.06149

4.6

82.83

19.5*

6.2

0.04163

35.4*

19.50

81.0*

25

0.00000

100.0*

0.00

100.0*

* Statistically significant

Validity criteria fulfilled:
yes
Conclusions:
The NOEC, EC10 and EC50 of MDIPA-Esterquat C16-18 and C18 unsatd. to algae in the presence of humic acid based on growth rate were 2.7, 3.2 (95% c.i. 2.5-4.1 mg/L) and 8.1 mg/L (95% c.i. 6.4-10 mg/L), respectively. The NOEC, EC10 and EC50 based on based on yield were 0.91, 1.3 (95% c.i. 0.45-3.7 mg/L) and 4.4 mg/L (95% c.i. 1.6-12 mg/L), respectively.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to MDIPA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0 (control), 0.25, 0.80, 2.5, 8.0 and 25 mg/L, corresponding to measured (TWA) 0.29, 0.91, 2.7, 6.2 and 25 mg/L in the presence of 4 mg/L humic (<2 mg/L DOC) acid under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006; Annex 5 corrected 28 July 2011).

Microscopic observations revealed no abnormalities.

The NOEC, EC10 and EC50 based on growth rate were 2.7, 3.2 (95% c.i. 2.5-4.1 mg/L) and 8.1 mg/L (95% c.i. 6.4-10 mg/L), respectively.

The NOEC, EC10 and EC50 based on based on yield were 0.91, 1.3 (95% c.i. 0.45-3.7 mg/L) and 4.4 mg/L (95% c.i. 1.6-12 mg/L), respectively.

All effect levels are given based on measured (TWA) concentrations.

 

 

Results Synopsis

 

Test Organism:Pseudokirchneriella subcapitata, strain NIVA CHL 1

Test Type: Static

 

72hr NOErC: 2.7 mg a.i./L

72 hr ErC10: 3.2 mg a.i./L                  95% c.i. 2.5 -4.1 mg/L

72 hr ErC50: 8.1 mg a.i./L                    95% c.i. 6.4 -10 mg/L

72hr NOEyC: 0.91

72 hr EyC10: 1.3 mg a.i./L                  95% c.i. 0.45 -3.7 mg/L

72 hr EyC50: 4.4 mg a.i./L                 95% c.i. 1.6 -12 mg/L

Endpoint(s) Effected:  growth rate, yield

Description of key information

72 h NOEC (growth rate) = 2.7 mg/mL, 72 h ErC10 = 3.2 (95% c.i. 2.5-4.1 mg/L), 72 h ErC50 = 8.1 mg/L (95% c.i. 6.4-10 mg/L);

72 h NOEC (biomass) = 0.91 mg a.i./mL, 72 h EyC10 = 1.3 (95% c.i. 0.45-3.7 mg/L), 72 h EyC50 = 4.4 mg/L (95% c.i. 1.6-12 mg/L)

(Pseudokirchneriella subcapitata, strain NIVA CHL 1); OECD guideline 201, static, GLP, RL1; in the presence of 4 mg/L humic acid

Key value for chemical safety assessment

EC50 for freshwater algae:
8.1 mg/L
EC10 or NOEC for freshwater algae:
3.2 mg/L

Additional information

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to MDIPA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0 (control), 0.25, 0.80, 2.5, 8.0 and 25 mg/L, corresponding to measured (TWA) 0.29, 0.91, 2.7, 6.2 and 25 mg/L in the presence of 4 mg/L humic acid (<2 mg/L DOC) under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006; Annex 5 corrected 28 July 2011).

Microscopic observations revealed no abnormalities.

The NOEC, EC10 and EC50 based on growth rate were 2.7, 3.2 (95% c.i. 2.5-4.1 mg/L) and 8.1 mg/L (95% c.i. 6.4-10 mg/L), respectively.

The NOEC, EC10 and EC50 based on based on yield were 0.91, 1.3 (95% c.i. 0.45-3.7 mg/L) and 4.4 mg/L (95% c.i. 1.6-12 mg/L), respectively.All effect levels are given based on measured (TWA) concentrations.

 

In a 96 h algal toxicity study conducted according to a modification of EPA OTS 797.1050 (Algal Toxicity, Tiers I and II), Selenastrum capricornutum was exposed MDEA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0, 2, 4, 8, 16 and 32 mg/L under static conditions. The 96 hr ErC50 value was 2.9 mg/L ( 95% confidence limit <2.0 - 4.2 mg/L; nominal).

This toxicity study is classified as not reliable and does not satisfy the guideline requirements for aquatic toxicity to algae (validity Klimisch 3). Deviations were: no analytical determination of test substance, river water used instead of algal media, and cell counts only done on controls at 0 h timepoint.