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:
from 2019-07-29 to 2019-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 201 and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
Samples were analyzed with an LC-MS method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling schedule
All concentration levels and the control were analytically verified via LC-MS at the start (0 hours) of the exposure with algae.

Sampling and pre-treatment
At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solution, media preparation
80.0 mg test item/L was freshly prepared with dilution water. The dispersion was treated with ultrasound for 5 minutes at room temperature. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Preparation of the test item solutions
Out of the stock solution five concentrations were tested in a geometric series with a dilution factor of 2.5: 2.05 - 5.12 - 12.8 - 32.0 - 80.0 mg/L.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

pH control
Three replicates of the highest test concentration, adjusted with 1M NaOH to pH 8.1 ± 0.2 (actual: 8.08), were tested under the test conditions as the test vessel.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Not specified
Test temperature:
min.: 21.0 max.: 22.0 mean value: 21.5
pH:
Geometric mean ,measured test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
89.7 (pH control) 8.08 8.13
88.0 5.84 7.12
36.2 7.74 8.25
13.9 8.07 8.35
5.53 8.22 8.47
2.05 8.23 8.55
Control 8.21 8.67
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal test item concentrations 2.05 - 5.12 - 12.8 - 32.0 - 80.0 mg/L
Geometric mean measured test item concentrations 2.05 – 5.53 – 13.9 – 36.2 – 88.0 (89.7 pH-control) 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 2 - 5 x 10^3 cells/mL, Actual: mean 5416 cells/mL
- Control end cells density: Mean 1629840 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:

Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm)
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 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: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, measured: 4846 - 6442 lux with a mean of 5607 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the concentration of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
55.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 36.4 - 88.0
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
65.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 36.9 - 88.0
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
82.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 37.8 - 88.0
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 2.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other:
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
40.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: CI: 10.8 - 87.6
Remarks:
without adjustment of pH
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
66.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: CI: 38.2 - 87.8
Remarks:
without adjustment of pH
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
89.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
with adjustment of pH
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 89.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
with adjustment of pH
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
89.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
with adjustment of pH
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 89.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
with adjustment of pH
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: The test media were clear throughout exposure period (possible turbidity related to algae growth not taken into account).
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2019-03-05 to 2019-03-08. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.

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 0.701 0.752 ± 0.534
95% confidence interval 0.660 – 0.750
Yield inhibition
EyC50 0.353 0.405 ± 0.308
95% confidence interval 0.312 – 0.394
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC10-,EC20- and EC50- values with confidence intervals of growth rate inhibition and the EC50- value for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:
For the results without adjustment of pH:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done with a significance level of 0.05.
For the results with adjustment of pH:
two sample t-test (significance level 0.05).

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.
•ToxRat Version 3.3.0, ToxRat Solutions GmbH

Any other information on results incl. tables

Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities. All effect values given are based on the geometric mean measured test item concentration of Barbituric acid.

Cell Densities

Geometric mean measured test item concentration       Replicate       Cell density [cells/mL]

[mg/L]                     No.       0 hours       24 hours       48 hours       72 hours

89.7 (pH control)       1       5416       24138       202911       1448905

                              2       5416       23097       174988       1350328

     3       5416       25596       192337       1326134

                         Mean       5416       24277       190079       1375122

88.0                        1       5416       4727       13419       52881

                            2       5416       5771       11484       55722

                              3       5416       5852       14247       46110

                        Mean       5416       5450       13050       51571

36.2                        1       5416       21631       174112       1295855

                              2       5416       23776       181842       1361865

                              3       5416       26757       217155       1384550

                        Mean       5416       24055       191036       1347423

13.9                      1       5416       20207       187362       1318442

                             2       5416       25660       163743       1471346

                              3       5416       20840       153578       1062142

                        Mean       5416       22236       168228       1283977

 5.53                       1       5416       27806       234455       1565347

                            2       5416       21544       188979       1528594

                            3       5416       24659       165310       1418041

                        Mean       5416       24670       196248       1503994

 2.05                      1       5416       27288       189358       1337135

                              2       5416       22763       187450       1405336

                              3       5416       28473       180216       1547336

                        Mean       5416       26175       185675       1429936

Control                    1       5416       28118       231827       1763085

                              2       5416       27904       246070       1943785

      3       5416       25338       210592       2001617

                              4       5416       21566       163665       1470421

                              5       5416       21013       153111       1355682

                              6       5416       23739       163470       1244449

                        Mean       5416       24613       194789       1629840

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration       Replicate       Growth rate       Inhibition of growth rate       Yield       Inhibition of yield

[mg/L]       No.       [d-1]       [%]       [cells/mL]       [%]

89.7 (pH control)       1              1.86       2              1443489       11

      2              1.84       3              1344912       17

      3              1.83       3              1320718       19

      Mean              1.85       3              1369706       16

88.0       1              0.76       60              47465       97

      2              0.78       59              50306       97

      3              0.71       62              40694       97

      Mean       (+)       0.75       60       (+)       46155       97

36.2       1              1.83       4              1290439       21

      2              1.84       3              1356449       17

      3              1.85       3              1379134       15

      Mean       (-)       1.84       3       (-)       1342007       17

13.9       1              1.83       3              1313026       19

      2              1.87       2              1465930       10

      3              1.76       7              1056726       35

      Mean       (-)       1.82       4       (-)       1278561       21

 5.53       1              1.89       0              1559931       4

      2              1.88       1              1523178       6

      3              1.86       2              1412625       13

      Mean       (-)       1.88       1       (-)       1498578       8

2.05       1              1.84       3              1331719       18

      2              1.85       2              1399920       14

      3              1.88       1              1541920       5

      Mean       (-)       1.86       2       (-)       1424520       12

Control       1              1.93                     1757669       

      2              1.96                     1938369       

      3              1.97                     1996201       

      4              1.87                     1465005       

      5              1.84                     1350266       

      6              1.81                     1239033       

      Mean              1.90                     1624424       

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       1.65       2.11       2.03       1.93       0.247       12.8       18.2

      2       1.64       2.18       2.07       1.96       0.284       14.5       

      3       1.54       2.12       2.25       1.97       0.377       19.1       

      4       1.38       2.03       2.20       1.87       0.430       23.0       

      5       1.36       1.99       2.18       1.84       0.431       23.4       

      6       1.48       1.93       2.03       1.81       0.294       16.2       

                    Mean       1.90       

                    SD ±       0.07       

                    CV [%]       3.47       

SD = Standard deviation       CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Barbituric acid was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the geometric mean measured test item concentration):
The test item lowered the pH value of the test solutions at concentrations of nominal 32 and 80 mg/L. The comparison of the results of treatments with and without adjustment of the pH showed that toxicity is only based on the influence of the pH.
With adjustment of pH: The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 89.7 mg/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 89.7 mg/L.
Executive summary:

The toxicity of Barbituric acid (Batch no.: JK20180906) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2019-07-29 to 2019-08-01 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5416 cells/mL. A stock solution with a nominal concentration of 80.0 mg test item/L was prepared with dilution water (see Table 2). The dispersion was treated with ultrasound for 5 minute at room temperature. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Out of the stock five concentrations were tested in a geometrical series with a dilution factor of 2.5: 2.05 - 5.12 - 12.8 - 32.0 - 80.0 mg/L, corresponding to the geometric mean measured test item concentrations of: 2.05 – 5.53 – 13.9 – 36.2 – 88.0 (89.7 pH-control) mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Additionally, three replicates of the highest test concentration, adjusted with 1M NaOH to pH 8.1 ± 0.2, was tested under the same test conditions as the test vessels. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. The concentrations of the test item Barbituric acid in all concentration levels and the control were analytically verified by LC-MS at the start and at the end of exposure.

The measured concentrations of the test item Barbituric acid in the fresh media were in the range 98 to 120% of the nominal values at the start of the exposure (0 hours) and between 102 and 112% of the nominal values at the end of the exposure (72 hours).

The test item lowered the pH value of the test solutions at concentrations of nominal 32 and 80 mg/L. The comparison of the results of treatments with and without adjustment of the pH showed that toxicity is only based on the influence of the pH.

NOEC, LOEC and ECx-values and 95% Confidence Intervals of Barbituric acid (0 - 72 hours)

based on the geometric mean measured test item concentration [mg/L]

      With adjustment of pH       Without adjustment of pH

      Inhibition of Growth Rate [mg/L]       Inhibition of Growth Rate [mg/L]

NOEC          89.7                                   36.2

LOEC       > 89.7                                   88.0

ErC10       > 89.7                                   55.4 (36.4 - 88.0)

ErC20       > 89.7                                   65.1 (36.9 - 88.0)

ErC50       > 89.7                                   82.5 (37.8 - 88.0)

      Inhibition of Yield [mg/L]       Inhibition of Yield

[mg/L]

NOEC          89.7                                   36.2

LOEC       > 89.7                                   88.0

EyC10       < 89.7                                   < 2.05

EyC20       > 89.7                                   40.6 (10.8 - 87.6)

EyC50       > 89.7                                   66.3 (38.2 - 87.8)