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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:
17 July 2018 - 20 July 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
GUIDELINE OECD 201 (2006, corrected 28 July 2011)
Deviations:
no
GLP compliance:
no
Remarks:
This study has not been performed according to GLP principles because based on the range-finding test,geometric mean concentration at the highest concentration was lower than the solubility of the test item henceforth test OECD No. 201 was not adapted.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number : 1700001162
- Expiration date of the lot/batch:12 June 2020
- Purity test date: January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in Darkness at a temperature of 20°C
- Stability under test conditions: The test material was not stable under test conditions (even without
algae) , measured concentrations varied by more than 20% from nominal concentrations at the end of the test and the geometric mean concentration at the highest concentration was lower than the solubility of the test material.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Since the test item is poorly soluble under the test conditions and according to the OECD 23, the choice of a solvent was necessary to solubilize the test material.A stock solution in dimethyl sulfoxide was prepared and test solutions were prepared by dilution of this solution.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The Test material was applied as liquid after solubilization in dimethyl sulfoxide (DMSO)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Based on the results of the range-finder, concentrations were prepared in OECD medium at 3 rates equivalent to: 0.1; 0.5 and 2 mg test item.L-1
- Sampling method: Test item solution and control were sampled and immediately analysed at 0h, 24h, 48h, and 72h.
- Sample storage conditions before analysis: No storage before analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dilution of the stock solution prepared by dissolving 200 mg of test material in 10 mL of dimethyl sulfoxide(DMSO), concentration of stock solution = 20 g/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):Dimethyl Sulfoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): according to the OECD monograph 23, the solvent concentration was not exceeded 0.1 mL.L-1 in the test and control solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater green Algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): CCAP (Culture Collection of Algae and Protozoa),
reference CCAP 1405/1.
- Method of cultivation:A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 to 4 days.

ACCLIMATION
- Culturing media and conditions (same as test or not):A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 to 4 days.
Medium: OECD medium
Temperature: 23 ± 2°C
Light: continuous illumination between 4440 and 8880 lux
pH: 7,80
Any deformed or abnormal cells observed: No abnormal appearance of the algae at the start and at the end of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Microscopic observation:
No abnormal appearance of the algae at the start and at the end of the test.
Test temperature:
0h (24.125 (°C)) ; 24h (24.125(°C)) ; 48h (24.125 (°C)) ; 72h ( 23.562 (°C))
pH:
pH : 0h Control (7.76) ; Solvent control (7.78) ; Test material at [0.1] (7.78) ; Test material at [0.5] (7.73) ; Test material at [2] (7.8)

pH : 72 h Control (7.53) ; Solvent control (7.63) ; Test material at [0.1] (7.45) ; Test material at [0.5] (7.45) ; Test material at [2] (7.47))
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type : open
- Material, size, headspace, fill volume:Glass Erlenmeyer flasks of 250 mL. .
- Initial cells density: 5 000 cells per millilitres..
- Control end cells density: 1453533 cells per millilitres.
- No. of organisms per vessel: 5 000 cells per millilitres.
- No. of vessels per concentration (replicates): 3 replicates for each
treatment

GROWTH MEDIUM
- Standard medium used: yes (OECD test medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: It was reconstituted with chemical reagents and ultra-pure water

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Between 4440 and 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman coulter Multisizer 3

TEST CONCENTRATIONS
- Test concentrations: 0.1, 0.5 and 2 mg test item.L-1 (nominal concentration).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate results: Starting date :18 May 2018 CEt50 (mg.L-1) :1,07 95% confidence limits (mg.L-1) : 0,94 – 1,2
Key result
Duration:
72 h
Dose descriptor:
other:
Remarks:
Test item was not stable under test conditions (measured concentrations varied by more than 20% from nominal concentrations at the end of the test), test OECD No. 201 is not adapted for this test item and it is not possible to estimate the LOEC,NOEC orECx
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
not specified
Remarks on result:
not determinable because of methodological limitations
Remarks:
The test item was not stable under test conditions (measured concentrations varied by more than 20% from nominal concentrations at the end of the test). Furthermore, the concentration of test item decreases significantly more with light than in the dark. The test item seems to be degrading intrinsically quickly in the OECD 201 test conditions.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):No abnormal appearance of the algae at the start and at the end of the test.
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: 1,07 (mg.L-1) with 95% confidence limits (mg.L-1) between 0,94 – 1,2

Validity criteria:



After 72 hours:

   Actul value  Reference value Compliance 
 Growth multiplication factor in the control within the 72-hour test period  291  > 16 Yes 
 Coefficient of variation of the average specific growth rates in the control during the whole test period  4.28  < 7  Yes
 Mean coefficient of variation for section-by-section specific growth rates (days 0 -1, 1 -2 and 2 -3) in the control  22.05  < 35  Yes

Validity criteria are met.

Quality control:

   Actual value  Reference value  Compliance
 Recovery at 20%  89  70 -120 Yes 
 Recovery at 80%  90  70 -120  Yes
 Within reproducibility relative standard deviation (recovery at 20%)  4.5  < 20  Yes
 Within reproducibility relative standard deviation ( recovery at 80%)  4.2  < 20  Yes
 Recoveries diffirence (%) between 20% and 80%  1  < 20  Yes

The analytical series are valid since the measured values are included in the reference interval.


Analytical results :

    Treatment ID  C01  C02  C03  C04  C05
    Target concentration [μg test item.L-1]

 0

(solvent)

 0

(solvent control)

 100  500  2000
0h analysed concentration [μg test item.L-1]             With algae  0 (< LOQ)

 0.034

(< LOQ)

 66.936  342.653  1496.431
 Deviation from target rate [%]  NA  NA  -33.06  -33.47  -25.18
 Blank (Without algae)

 0.11

(<LOQ)

 0.870

(<LOQ)

68.728   333.306  1612.583
 Deviation from target rate [%]  NA  NA  -31.27  -33.34  -19.37
72h analysed concentation [μg test item.L-1]            With algae  0.044(<LOQ)

0.004

(<LOQ)

 0.020

(<LOQ)

0.222

(<LOQ) 

 116.903
 Deviation from target rate [%]

 NA

 NA  -99.98  -99.96  -94.15
 Blank (Without algae)

 0.033

(<LOQ)

0.005

(<LOQ)

0.001

(<LOQ) 

 0.444

(<LOQ)

 102.764
 Deviation from target rate[%]  NA  NA  -100  -99.1  -94.86
         Geometric mean concentration after 72h [μg test item.L-1]  With algae  NA  NA  1.157  8.726  418.255
 Deviation from target rate[%]  NA  NA  -98.84  -98.25  -79.09
 Blank (Without algae)  NA  NA

 0.195

(<LOQ)

 12.171

 407.081

 Deviation from target rate[%]  NA  NA  -99.80  -97.57  -79.65

NA = not applicable.LOQ = limit of quantification = 1 μg active substance.L-1

According to the table above, the test item was not stable under test conditions (even without

algae) and the geometric mean concentration at the highest concentration was lower than the

solubility of the test item.

Then, the highest concentration was prepared again, maintained at 22,5°C and 8700 lux and analysed after 0, 24 and 48h, in order to try to obtain a concentration higher or equal to the solubility of the test item

Results with algae are the following:

 Target concentration[μg test item.L-1]  2000
 0h analysed concentration [μg test item.L-1] 1959.867 
 Deviation from target rate [%] -2.01 
 24h analysed concentration [μg test item.L-1] 385.511 
 Deviation from target rate [%]  -80.72
 48h analysed concentration [μg test item.L-1]  151.833
 Deviation from target rate [%]  -92.41
 Geometric mean concentrations [μg test item.L-1] 485.896 
 Deviation from target rate [%]  -75.71

Geometric mean concentration at the highest concentration was still lower than the solubility of the test item given by the sponsor after day by day analysis.

Another test was performed without algae in dark and in the light in the same conditions of the

test in a thermostatic room. The test solutions of test item at 2 mg.L-1 in the OECD medium

were analysed each day, and the results are the following:

 μg.L-1  J0  J1  J2  J3
 

Test item at 2000 μg.L-1-

Light_01

 3059.79  629.25  255.64  2.92
 

Test item at 2000 μg.L-1-

Light_02

 3144.96 488.63   308.56 33.46 

Test item at 2000 μg.L-1-

Dark_01

 
2940.63  2854.82   1520.83 10.10 
 

Test item at 2000 μg.L-1-

Dark_02

3075.07  2761.14   1561.35 17.19 

The concentration of test item decreases significantly more with light than in the dark.

Since the test item was not stable under test conditions (measured concentrations varied by more than 20% from nominal concentrations at the end of the test), test OECD No. 201 is not adapted for this test item and it is not possible to estimate the LOEC, NOEC or ECx.


Validity criteria fulfilled:
yes
Conclusions:
The effects of 2-(hexadecanoylamino)acetic acid diluted in OECD test medium at the rates of
0.1, 0.5 and 2 mg test item.L-1 to Pseudokirchneriella subcapitata were investigated under
laboratory conditions.

The study is valid since the growth multiplication factor in the control within the 72-hour test
period was higher than 16 (actual value: 291), the coefficient of variation of the average specific
growth rates in the control was less than 7% (actual value: 4.28%) and the mean coefficient of
variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was
less than 35% (actual value: 22.05% after 72h).
Test item was not stable under test conditions (measured concentrations varied by more
than 20% from nominal concentrations at the end of the test).
The geometric mean concentration at the highest concentration was lower than the solubility of the test item.
Furthermore, the concentration of test item decreases significantly more with light than in the
dark. The test item seems to be degrading intrinsically quickly in the OECD 201 test conditions.
So, the test OECD No. 201 seems to be not adapted for this test item and it is not possible to
estimate the LOEC, NOEC or ECx.
Executive summary:

The objective of this study was to assess the effects of test item 2-(hexadecanoylamino)acetic acid on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201.

Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201. Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl sulfoxide was prepared. Test solutions were prepared by dilution of this solution.

Test item quantification was performed according to a validated analytical method (final report RRCo-000154_01) LC-MS/MS from Sciex (LC Sciex Exion AD and MS QTRAP 5500) as mass spectrometry detection to measure 2-(hexadecanoylamino)acetic acid concentrations at 0, 24, 48 and 72 hours.

The effects of 2-(hexadecanoylamino)acetic acid diluted in OECD test medium at the rates of 0.1, 0.5 and 2 mg test item.L-1 to Pseudokirchneriella subcapitata were investigated under laboratory conditions.

The study is valid since the growth multiplication factor in the control within the 72-hour test period was higher than 16 (actual value: 291), the coefficient of variation of the average specific growth rates in the control was less than 7% (actual value: 4.28%) and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was less than 35% (actual value: 22.05% after 72h).


The geometric mean concentration at the highest concentration was lower than the solubility of the test item.Furthermore, the concentration of test item decreases significantly more with light than in the dark.


Since the test item was not stable under test conditions (measured concentrations varied by more than 20% from nominal concentrations at the end of the test), test OECD No. 201 is not adapted for this test item and it is not possible to estimate the LOEC, NOEC or ECx. 

Description of key information

According to OECD 201.Guideline, to assess the effects of test item 2-(hexadecanoylamino)acetic acid on the growth of Pseudokirchneriella subcapitata over a period of 72 hours :

Based on the results of the range-finding test, the geometric mean concentration at the highest concentration was lower than the solubility of the test item and it seems that the test item degrade intrinsically quickly in the OECD 201 test conditions.Furthermore, the concentration of test item decreases significantly more with light than in the dark.


Since the test item was not stable under test conditions (measured concentrations varied by more than 20% from nominal concentrations at the end of the test), test OECD No. 201 is not adapted for this test item and it is not possible to estimate the LOEC, NOEC or ECx. 

Key value for chemical safety assessment

Additional information