Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-27 - 2016-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23-Mar-2006
Deviations:
yes
Remarks:
The concentration of NaHCO3 was increased to be twice of that indicated in the Guideline 201. This concentration has been found to be optimal. This deviation is not considered to have an influence on the integrity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Other: Storage conditions: Ambient temperature

Sampling and analysis

Analytical monitoring:
yes
Remarks:
TOC
Details on sampling:
- Concentrations (nominal with measured in brackets): 1.25 (no net TOC), 2.5 (no net TOC), 5.0 (0.14), 10.0 (0.76), 20 (3.45) mg/L
- Sampling method: The supporting analyses could not be performed on the same day of collection of the specimens.
Therefore, these specimens from freshly prepared test solutions were stored at 2-8°C and then analysed. Further aliquots of the treatments (t0 and t72hr) were stored deep-frozen for control purpose later-on.

Test solutions

Vehicle:
no
Remarks:
medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aliquot of the algal stock culture was diluted with test medium to an absorbance value of E578 nm = 0.015 of the algal suspension giving an initial algal cell concentration of 3*103 cells/mL. The determination of cell concentration was performed by photometrical measurement using a calibration curve prepared by using a spectrophotometer at 578 nm. The test item was prepared as follows: Separate solutions (see below) were made for each test concentration in ultrapure water. Aliquots of these solutions were made up with „intermediate dilution“ (see section above) and algal inoculum (or diluted „intermediate dilution“) to give 50 mL of final volume.
Prior to use in the test the test item was milled in a mortar in order to get fine particles of the test item which could be better dissolved in the aqueous phase hereafter. The test item was introduced into the test solutions as follows: First, suspensions of the test item were individually prepared for each test concentration in ultrapure water and stirred on a magnetic stirrer for 60 min. (screening test) to ≈24 hr (definitive main test) at room temperature after 3 to 5*11 minutes of ultrasonication. Thereafter the suspensions were transferred into 8 test vessels (screening test) or 10 test vessels, respectively (main test). By the addition of the "intermediate dilution" and the algal inoculation the final nominal concentrations (see below) were achieved.
For the inoculated test solutions with the test item, as well as those without any test item, seven replicates for the main test and five replicates for the screening tests were incubated under constant light. As a reagents’ Blank without algal inoculation, three test solutions were prepared as described above containing a 1:10 diluted intermediate dilution instead of the algal pre-culture. These blanks were incubated under the same conditions under constant light as the test solution to be tested. The measured turbidity in those test solutions was subtracted from those with the algal inoculation. In the same way, seven control solutions without any test item but with algal inoculation were prepared within the main test as well as one test solution without any test item and without algal inoculation.
- Controls: medium without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Strain-No. 86.81

ACCLIMATION
- Acclimation period / culturing media and conditions (same as test or not): Three days prior to starting the test, a pre-culture was inoculated using the algal stock culture. This culture was prepared in „pre-culture medium“ using 100 mL of the Mineral Nutrient Salts stock solution and 10 mL of each of FeCl3- Solution and Trace Elements stock solutions and 1 g NaHCO3 in 880 mL ultrapure water. This "intermediate dilution" was used for the pre-culture at a final concentration of 50 mL/500 mL prepared in ultrapure water.
- Any deformed or abnormal cells observed: none stated

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
23.1-24.2 °C (measured by aid of a data logger, system ELPRO)
pH:
8.18 - 9.32 during 1st main test, during 2nd main test 8.55 - 9.27 (screening: 7.91 - 9.16)
Nominal and measured concentrations:
Nominal: 1.25, 2.5, 5.0, 10.0, 20 mg/L
Found (t0): n.n., n.n., 0.14, 0.76, 3.45 mg/L
Found (t72h): n.n., 0.07, 0.32, 0.88, 2.43 mg/L
n.n. = no net TOC
Details on test conditions:
TEST SYSTEM
- Test vessel: cylinders
- Material, size, headspace, fill volume: 50 mL-glass cylinders, heat sterilized
- Initial cells density: 3*10E3/mL
- Control end cells density: 1.0*10E5 (2nd main test, at highest concentration of 20 mg/L)
- No. of vessels per concentration (replicates): 7 (main test) / 5 (screening)
- No. of vessels per control (replicates): 7 (main test) / 5 (screening)

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water generatingsystems: Purelab Classic D1, USF Seral; Ser.-No. 1470984-10 01 (ELGA Labwater)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: illumination rate of ≥ 120 µE/m²s ([≈8000 Lux]

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer at 578 nm and additionally a fluorimeter in the case there was a turbidity due to the test item in the treatments; at t0 (theoretical value on basis of the measured value for the inoculum), after 26 hours (definitive main test), 46.75 hours (definitive main test) and 72 hours

TEST CONCENTRATIONS
- Test concentrations (Range finding study, screening): 1, 10, 100 mg/L
Reference substance (positive control):
no
Remarks:
A reference item was not tested within the biological part of this study. Within the analytical part, the test item served as a reference item.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: nominal: 33.7 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: nominal: 4.8 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: nominal: 9.3 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: nominal: 2.5 mg/L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: nominal: 5.0 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 201 with no relevant deviations (only minor deviation with no influence on the integrity of the study), the results were so obtained via a scientifically reasonable method. The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. Hence, there is no doubt that the obtained results are not reliable: The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L (screening test), and the definitive main test was performed using five concentrations in the range of 1.25 to 20 mg/L.
As a conclusion of the analytical part of this study, it can be stated that the concentrations of Octadecanoic acid, reaction products with tetraethylenepentamine at concentration steps around the EC50 remained sufficiently stable during incubation of 72hr: after 72 hr of incubation recoveries of >97 % of the initial values measured at time t0 were obtained in the treatments at concentrations around the EC50. At lower concentrations it is assumed that a certain amount of the test item may have been adsorbed to surfaces as minor concentrations were found. Based on this, the reported effect concentrations (EC values) should refer to measured concentrations of the test item.
The following EC-values (72h; growth rate; measured concentrations) were determined accordingly:
LOEC 0.18 mg/L
NOEC 0.03 mg/L
EC50 = 7.02 mg/L
Based on these results, the test item does not need to be classified as aquatic acute Cat. 1 (EC50 <1mg/L), M factor 1. Further, with regard to chronic toxicity and taking into account the facts that the test item is soluble in water and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 2 based solely on the results of this study. As the results of the OECD 202 are more critical, the classification should be based on the results with daphnids.
Executive summary:

The toxicity of Octadecanoic acid, reaction products with tetraethylenepentamine towards algae was tested according to OECD-Guideline No. 201 (GLP), in the Version dated 23-March-2006. The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L (screening test ans 1st main test), and the definitive main test was performed using five concentrations in the range of 1.25 to 20 mg/L.The following EC-values were obtained:

On the Basis of the nominal Concentrations [mg test item/ L]

 Yield (0 - 72 hr)                                

                             EC10           2.3

           95%-CL     lower           2.2

                            upper           2.4             

                             EC20           3.4

           95%-CL     lower           3.3

                            upper           3.5             

                             EC50           6.9

           95%-CL     lower           6.6

                            upper           7.2             

                Yield    LOEC           5.0

                           NOEC           2.5

Section-by-section growth rate (48.25-72hr)

                             EC10           7.0

           95%-CL     lower           5.4

                            upper           9.2             

                             EC20           8.9

           95%-CL     lower           6.9

                            upper            11.7 

                             EC50          14.2

           95%-CL     lower           10.2

                            upper           19.6

                       

                            LOEC           10.0

                           NOEC           5.0

Growth rate (0 - 72 hr)                        

                            EC10           4.8

           95%-CL     lower           3.2

                            upper           7.1 

                             EC20           9.3

           95%-CL     lower           6.1

                            upper           14.2

                             EC50          33.7

           95%-CL     lower          17.8

                            upper            61.6

      Growth rate    LOEC           5.0

                           NOEC           2.5

On the Basis of the measured Concentrations [mg test item/ L]

 Yield (0 - 72 hr)                                

                             EC10           0.04

           95%-CL     lower           0.02

                            upper           0.09             

                             EC20          0.09

           95%-CL     lower           0.05

                            upper           0.18             

                             EC50           0.36

           95%-CL     lower           0.16

                            upper           0.82             

                Yield    LOEC           0.18

                           NOEC           0.03

Section-by-section growth rate (48.25-72hr)

                             EC10           0.35

           95%-CL     lower           0.20

                            upper           0.61             

                             EC20           0.54

           95%-CL     lower           0.32

                            upper            0.95 

                             EC50          1.30

           95%-CL     lower           0.66

                            upper           2.53

                       

                            LOEC           0.69

                           NOEC           0.18

Growth rate (0 - 72 hr)                        

                            EC10           0.17

           95%-CL     lower           0.07

                            upper           0.38 

                             EC20           0.60

           95%-CL     lower           0.25

                            upper           1.46 

                             EC50          7.02

           95%-CL     lower           1.81

                            upper            25.12

      Growth rate    LOEC           0.18

                           NOEC           0.03

As a conclusion of the analytical part of this study, it can be stated that the concentrations of Octadecanoic acid, reaction products with tetraethylenepentamine at concentration steps around the EC50 remained sufficiently stable during incubation of 72hr: after 72 hr of incubation recoveries of >97 % of the initial values measured at time t0 were obtained in the treatments at concentrations around the EC50. At lower concentrations it is assumed that a certain amount of the test item may have been adsorbed to surfaces as minor concentrations were found. Based on this, the reported effect concentrations (EC values) should refer to measured concentrations of the test item.

The validity criteria for the study as given by the OECD Guideline 201 were all met and the study is therefore considered to be valid.