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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-06-22 to 2010-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted under GLP and following OECD guideline. Experiment and results are well described.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not relevant
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 4.6, 10, 22, 46, and 100 mg/L
- Sampling method: 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 3 mL
At the end of the exposure period, the replicates with algae from each concentration were pooled before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/l applying 15-30 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: P. subcapitata
- Strain: NIVA CHL1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): no data
- Method of cultivation: Algae 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
- Acclimation period: 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): Culture media used was M2 medium (previously described in Details on test solutions).
- Any deformed or abnormal cells observed: no data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
0.24 mmol/L (i.e. 24 mg CaCO3/L)
Test temperature:
21-24°C
pH:
8.1 +/- 0.2
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
- nominal concentrations: 4.6, 10, 22, 46, and 100 mg/L
- measured concentrations: 3.9, 8.8, 21, 41, and 91 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 50 mL of test solution
- Aeration:no data
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 187.2 x 10^4 cells/mL
- No. of organisms per vessel: 5 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 + 1 extra replicate of the control and each test concentration for sampling purposes; 2 extra replicates of the highest concentration without algae.
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/l
K2HPO4.3H2O 52 mg/l
MgSO4.7H2O 75 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.2H2O 35 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
(NH4)6Mo7O24.4H2O 0.018 mg/l

Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: no data
- Photoperiod: 24:0
- Light intensity and quality: 90-103 µE.m-².s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: couting chamber and microscope at the beginning of the experiment, then spectrophotometer. At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank. Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Chlorophyll measurement: no
- Other: none.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 4.6, 10, 22, 46, and 100 mg/L
- Results used to determine the conditions for the definitive study: The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 presents the percentages growth rate reduction and yield inhibition per concentration. The combined limit/range-finding test did not meet one of the validity criteria, i.e. the coefficient of variation of average specific growth rates in the control was too high. Despite this, the test could be used as a range-finder for the final test. Samples taken from nominal 10 mg/l were analysed and showed an initial concentration of 9.6 mg/l. This concentration remained stable during the first 24 hours of exposure (95% of initial) and slightly decreased towards the end of the tes. Table 1 and 2 are presented in "Any other information on materials and methods incl. tables."
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 68 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 55-82 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 17 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 13-22 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and biomass
Details on results:
no data
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: EC50(72h) for growth rate reduction = 1.1 mg/L
EC50(72h) for yield inhibition = 0.59 mg/L
- Other: none
Reported statistics and error estimates:
none

Table 3: Individual cell densities

Number of inoculated cells at t=0: 1x104cells/ml

 

 

 

 

 

 

 

 

Average conc.

Vessel

Exposure time (hours)

TERPINEOL

number

 

 

 

 

mg/l

 

0

24

48

72

control

1

1.00

6.99

36.81

182.64

 

2

1.00

6.41

37.93

199.10

 

3

1.00

6.54

33.65

186.62

 

4

1.00

6.75

37.27

167.86

 

5

1.00

6.34

38.28

198.86

 

6

1.00

6.90

36.77

188.08

3.9

1

1.00

6.38

34.28

194.50

 

2

1.00

6.91

40.52

196.46

 

3

1.00

6.93

38.68

206.26

8.8

1

1.00

5.33

27.62

145.20

 

2

1.00

5.72

29.15

129.52

 

3

1.00

6.37

27.53

125.28

21

1

1.00

5.45

21.82

73.19

 

2

1.00

4.78

19.83

83.82

 

3

1.00

4.95

24.28

91.66

44

1

1.00

4.17

14.16

20.98

 

2

1.00

4.00

10.06

27.17

 

3

1.00

4.67

11.70

23.05

91

1

1.00

3.30

6.38

8.20

 

2

1.00

3.53

6.62

6.92

 

3

1.00

3.04

7.35

8.59

 

Table 4: Calculation of growth rate and yield

Average conc.

Vessel

Growth rate

Yield

Growth rate red.

Yield inhib.

TERPINEOL

number

(µ)

(x104cells/ml)

(%)

(%)

(mg/l)

 

0-72 h

0-72 h

0-72 h

0-72 h

control

1

0.07233

181.64

 

 

 

2

0.07353

198.10

 

 

 

3

0.07263

185.62

 

 

 

4

0.07115

166.86

 

 

 

5

0.07351

197.86

 

 

 

6

0.07273

187.08

 

 

 

mean

0.07265

186.19

 

 

 

CV

1%

 

 

 

3.9

1

0.07320

193.50

-1

-4

 

2

0.07334

195.46

-1

-5

 

3

0.07402

205.26

-2

-10

8.8

1

0.06914

144.20

5

23

 

2

0.06755

128.52

7

31

 

3

0.06709

124.28

8

33

21

1

0.05963

72.19

18

61

 

2

0.06151

82.82

15

56

 

3

0.06275

90.66

14

51

44

1

0.04227

19.98

42

89

 

2

0.04586

26.17

37

86

 

3

0.04358

22.05

40

88

91

1

0.02922

7.20

60

96

 

2

0.02687

5.92

63

97

 

3

0.02987

7.59

59

96

Table 5: Calculation of growth rate (section-by-section)

Average conc.

Vessel

Growth rate (µ)

Growth rate reduction (%)

TERPINEOL

number

 

 

 

 

 

 

(mg/l)

 

0-24 h

24-48 h

48-72 h

0-24 h

24-48 h

48-72 h

control

1

0.08102

0.06922

0.06674

 

 

 

 

2

0.07741

0.07408

0.06909

 

 

 

 

3

0.07825

0.06825

0.07138

 

 

 

 

4

0.07956

0.07119

0.06271

 

 

 

 

5

0.07695

0.07492

0.06865

 

 

 

 

6

0.08048

0.06972

0.06801

 

 

 

 

mean

0.07895

0.07123

0.06776

 

 

 

 

CV

2%

4%

4%

 

 

 

 

The mean CV for section-by-section specific growth rate was:

7%

 

 

3.9

1

0.07722

0.07006

0.07233

2

2

-7

 

2

0.08054

0.07370

0.06578

-2

-3

3

 

3

0.08066

0.07164

0.06974

-2

-1

-3

8.8

1

0.06972

0.06855

0.06915

12

4

-2

 

2

0.07267

0.06785

0.06214

8

5

8

 

3

0.07715

0.06099

0.06314

2

14

7

21

1

0.07065

0.05780

0.05043

11

19

26

 

2

0.06519

0.05928

0.06006

17

17

11

 

3

0.06664

0.06626

0.05535

16

7

18

44

1

0.05950

0.05094

0.01638

25

28

76

 

2

0.05776

0.03843

0.04140

27

46

39

 

3

0.06421

0.03827

0.02825

19

46

58

91

1

0.04975

0.02747

0.01046

37

61

85

 

2

0.05255

0.02620

0.00185

33

63

97

 

3

0.04633

0.03679

0.00650

41

48

90

 

Validity criteria fulfilled:
yes
Conclusions:
Effect of Terpineol multiconstituent was tested in an OECD guideline 201 experiment. The study met the acceptability criteria prescribed by the protocol and was considered valid. Terpineol multiconstituent reduced growth rate and inhibited the yield of this fresh water algae species significantly at 8.8 mg/L and higher. The EC50 for growth rate reduction (72h-ERC50) was 68 mg/L with a 95% confidence interval ranging from 55 to 82 mg/L. The EC50 for yield inhibition (72h-EYC50) was 17 mg/L with a 95% confidence interval ranging from 13 to 22 mg/L. The NOEC for both growth rate reduction and yield inhibition was 3.9 mg/L.
Executive summary:

Effect of Terpineol multiconstituent was tested in a fresh water growth inhibition test on algae, Pseudokirchneriella subcapitata, following OECD guideline 201, 2006. In addition, the procedures were designed to meet the test methods of Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 andthe ISO International Standard 8692, 2004.

Three replicates of exponentially growing algal cultures were exposed to nominal Terpineol concentrations of 4.6, 10, 22, 46 and 100 mg/L. The initial cell density was 104cells/mL and the total test period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

At the start of the test, the actual test concentrations were in agreement with nominal concentrations (96-101%). These concentrations remained stable during the first 24 hours of exposure (89-97% of initial concentrations) and slightly decreased towards the end of the test (77-92% of initial concentrations). Given these results, effect parameters were based on Time Weight Average exposure concentrations that were calculated to be 3.9, 8.8, 21, 44 and 91 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid.

Terpineol reduced growth rate and inhibited the yield of this fresh water algae species significantly at 8.8 mg/L and higher.

The EC50for growth rate reduction (72h-ERC50) was 68 mg/L with a 95% confidence interval ranging from 55 to 82 mg/L.

The EC50for yield inhibition (72h-EYC50) was 17 mg/L with a 95% confidence interval ranging from 13 to 22 mg/L.

The NOEC for both growth rate reduction and yield inhibition was 3.9 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
key study
Study period:
2016-03-07 to 2016-03-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Calculation method is used ; calculation method applicable for the endpoint.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
calculation method
Principles of method if other than guideline:
The acute toxicity to aquatic algae was determined using a validated QSAR for the Mode of Action in question. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study.
Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The ErC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF.
The method has been validated using data derived from 72-hour ErC50 tests on aquatic algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
not applicable
Test organisms (species):
other: algae spp.
Details on test organisms:
not applicable
Test type:
other: calculation method
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
72h-ErL50 (effective loading rate of WAF)
Post exposure observation period:
not applicable
Hardness:
Hardness is not a necessary component of the WAF calculation
Test temperature:
The Temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine EL50s for algae between 12 and 28°C.
pH:
The pH is not a necessary component of the WAF calculation
Dissolved oxygen:
The oxygen concentration is not a necessary component of the WAF calculation
Salinity:
Salinity is not a necessary component of the WAF calculation. However as the fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater organisms
Nominal and measured concentrations:
The calculation determines measured concentrations
Details on test conditions:
calculation method
Reference substance (positive control):
not required
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
19 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable

Table 6.1.5/1: Short-term toxicity to aquatic algae values for components used in the calculation.

constituents

72h-ErC50 (mg/L)

α-terpineol

62

γ-terpineol

62

β-terpineol

52

terpinolene

0.60

3-terpinen-1-ol

62

borneol

22

fenchol

22

terpinen-1-ol 4

62

limonene

0.50

Table 6.1.5/2: Expected concentrations at this 72h-ErL50.

constituents

concentration in the WAF (mg/L)

α-terpineol

12

γ-terpineol

1.9

β-terpineol

0.97

terpinolene

1.5

3-terpinen-1-ol

0.67

borneol

0.76

fenchol

0.46

terpinen-1-ol 4

0.30

limonene

0.08

Validity criteria fulfilled:
yes
Conclusions:
The 72h-ErL50 for Terpineol multconstituent is estimated at 17 mg test item/L.
Executive summary:

The registered substance is a multiconstituent with a well-defined composition for which the relative percentage and its reported variation of each constituent is known. Its acute toxicity to aquatic algae property has been investigated using an in-house calculation method that replaces an OECD 201 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions).

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study.

Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is conducted to remove this non-bioavailable fraction.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF.

The 72-h ErL50 was 17 mg test material/L. ErL50 were based on growth rate.

 

Description of key information

The substance exhibits an ErC50 for freshwater algae of 17 mg/L and a NOErC of 3.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
17 mg/L
EC10 or NOEC for freshwater algae:
3.9 mg/L

Additional information

The registered substance is a multiconstituent substance with a well-defined composition for which the relative percentage of each constituent is known. The ecotoxicological properties of the registered substance may be derived from the aquatic toxicity values of the individual components using an additivity calculation approach.

It has been observed that CLP additivity calculations for mode of action 1 (MOA 1) compounds are unreasonably conservative when compared to classic experimental studies using Water Accommodated Fractions (WAF), and this has been proved in a number of cases with substances of natural origin. Indeed, substance of natural origin may be composed of hydrophilic alcohol molecules and hydrophobic hydrocarbon terpene molecules. Therefore, when a WAF is performed most of substances fully dissolved in the aqueous phase are the hydrophilic fraction while the hydrophobic fraction (the more toxic elements for MOA 1 substances) may be below their water solubility value. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study. On the other hand, the CLP additivity approach is calculated on the basis that all the substances are at their maximum solubility. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction. These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation. The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading of the WAF.

A predicted acute toxicity value to Algae is available. The estimated EL50 for Daphnid is 19 mg/L. This prediction is obtained with QSAR for the Mode of Action 1, based on validated data derived from standard toxicity test, for which the concentrations of the test item had been determined by chemical analyses over the test period. The prediction is sufficiently robust, and is appropriate for the purposes of chemical safety assessment required for REACH.

Besides, one reliable experimental study is available for the registered substance. The toxicity to Daphnia magna was studied according to OECD TG No. 202 and GLP. Daphnids were exposed to substance at nominal test concentrations of 0 (test medium control), 4.6, 10, 22, 46 and 100 mg/L over a test period of 72 hours (static test). Concentrations were analytically verified and measured concentrations were 3.9, 8.8, 21, 41 and 91 mg/L. The endpoint was based on measured concentrations (time-weighted averaged). The 72-h ErC50 was 68 mg/L and the 72-h NOErC was 3.9 mg/L.