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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Terephthalaldehyde
EC Number:
210-784-8
EC Name:
Terephthalaldehyde
Cas Number:
623-27-8
Molecular formula:
C8H6O2
IUPAC Name:
benzene-1,4-dicarbaldehyde
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch (Lot) Number: 180401
Expiry date: 01 April 2020 (retest date)
Physical Description: Light yellow powder
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Sampling and analysis

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 2.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
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 checked by running a test vessel at nominally 10 mg/L 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.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Terephthaldehyde tested was a light yellow powder with a purity of 99.6% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of nominally 100 mg/L applying a three-day period of magnetic stirring to ensure complete dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
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.
Any residual volumes were discarded.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture 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.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
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 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 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 3 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.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water 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

Study design

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

Test conditions

Hardness:
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21-24°C.
pH:
pH 8.1 ± 0.2
Dissolved oxygen:
N.D.
Salinity:
N.D.
Conductivity:
N.D.
Details on test conditions:
Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a nominal concentration of 100 mg/L in the limit test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to nominally 0.10, 1.0 and 10 mg/L in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test, algae were not observed under a microscope to verify a normal and healthy appearance.

Final Test

Test Concentrations
Terephthaldehyde 1.0, 3.2, 10, 32 and 100 mg/L.
Control Test medium without test item or other additives.
Replicates 3 replicates of each test concentration,
6 replicates of the control,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium M2 (see paragraph 4.7.)
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 94 to 95 µE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.



Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 6.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 5.9 - <= 6.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.95 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 0.92 - <= 0.98 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Reported statistics and error estimates:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 188).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 12%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.87%).

Any other information on results incl. tables

Combined Limit/Range-Finding Test

The mean cell densities measured during the combined limit/range-finding test are presented inTable 1.n.d.: not determined

Table 2andTable 3present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

A dose-related increase of algal growth inhibition was observed at the three highest test concentrations at the end of the test, while no inhibition was recorded at the lowest test concentration.

Based on these results, samples taken from 1.0 and 10 mg/L were analysed. The measured concentrations at the start of the test were 0.97 and 9.9 mg/L, respectively. At the end of the test, the measured concentrations were at 1.0-46% of initial concentrations (see also Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1
Mean Cell Densities (x104Cells/mL) during the Combined Limit/Range-Finding Test

Time (h) 

Terephthaldehyde; Nominal conc. (mg/L)

Control

0.10

1.0

10

100

0

1.0

1.0

1.0

1.0

1.0

24

8.1

n.d.

n.d.

n.d.

2.4

48

57.1

n.d.

n.d.

n.d.

2.2

72

268.7

269.1

263.9

7.4

2.0

n.d.: not determined

Table 2
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

Terephthaldehyde
Nominal conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.864

0.0150

6

 

0.10

1.865

0.0136

3

0.0

1.0

1.859

0.0048

3

0.3

10

0.669

0.0130

3

64

100

0.221

0.0498

6

88

Table 3
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

Terephthaldehyde
Nominal conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

267.7

12.34

6

 

0.10

268.1

11.09

3

-0.2

1.0

262.9

3.82

3

1.8

10

6.4

0.29

3

98

100

1.0

0.29

6

100

 

 

             Final Test

          Measured Test Item Concentrations

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from all test concentrations and the control were analysed. The measured concentrationsat the start of the testwere 0.94, 3.1, 9.7, 30 and 97 mg/L at nominally 1.0, 3.2, 10, 32 and 100 mg/L, respectively. During the 72-hour exposure period, the initially measured concentration at nominally 1.0 mg/L had decreased below the Limit of Detection (i.e. LOD=0.0073 mg/L). All other concentrations were measured to be at 0.40 – 95% of initial concentrations at the end of the test.

The concentrations measured in the samples taken from solutions with algae were slightly lower than the concentrations measured in the samples without algae (seeTable 4). The presence of the algae thus may impact the stability of the test item in test medium throughout the test.

Based on the obtained results, Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters (seeTable 4).

Table 4
Time Weighted Average Versus Nominal Concentrations

Terephthaldehyde

Nominal conc. (mg/L)

Measured concentration (mg/L)

TWA conc. (mg/L)

t=0h

t=24h

t=72 h

1.0

0.939

0.66

0.00372

0.29

3.2

3.07

2.64

0.0123

1.1

10

9.71

8.90

4.62

7.4

101

9.85

9.77

7.29

8.91

32

30.3

29.9

22.5

27

100

97.0

96.2

92.1

95

1Without algae;
2Estimated as being half of the LOD (i.e. 0.0073*0.5 mg/L);
3Estimated by extrapolation of the calibration curve.

      Mean Cell Densities

Figure 1shows growth curves at different TWA concentrations of Terephthaldehyde. The individual and group mean cell densities measured at 24h intervals are given inTable 10.

  Inhibition of Growth Rate and Inhibition of Yield

Table 5shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereasTable 6shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized inTable 7(see Appendix 1 for the individual values). Statistical analysis of the data is shown inAppendix 2andAppendix 3.

A dose-related increase of algal growth rate inhibition was observed at all concentrations tested at the end of the test. Statistically significant inhibition of growth rate was found at TWA concentrations of 1.1 mg/L and higher.

A dose-related increase of yield reduction was observed at all concentrations tested at the end of the test. Statistically significant reduction of yield was found at TWA concentrations of 1.1 mg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration of 1.1 mg/L when compared to the control.

Table 5
Growth Rate and Percentage Inhibition for the Total Test Period

Terephthaldehyde
TWA conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.744

0.0151

6

 

0.29

1.743

0.0183

3

0.056

1.1

1.446

0.0116

3

17*

7.4

0.525

0.0366

3

70*

27

0.585

0.0193

3

66*

95

0.302

0.0354

3

83*

* Effect was statistically significant.

Table 6
Growth Rate and Percentage Inhibition at Different Time Intervals

 

Terephthaldehyde

TWA conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.954

 

1.755

 

1.524

 

0.29

3

2.007

-2.7

1.732

1.3

1.492

2.1

1.1

3

1.660

15

1.159

34

1.518

0.37

7.4

3

1.612

18

0.242

86

-0.278

118

27

3

1.188

39

0.315

82

0.253

83

95

3

0.798

59

0.252

86

-0.144

109


Table 7
Yield and Percentage Inhibition for the Total Test Period

Terephthaldehyde
TWA conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

186.6

8.22

6

 

0.29

186.1

10.41

3

0.28

1.1

75.6

2.65

3

59*

7.4

3.9

0.54

3

98*

27

4.8

0.34

3

97*

95

1.5

0.26

3

99*

* Effect was statistically significant.

  Determination of Effect Concentrations

Table 8shows the effect parameters based on TWA concentrations, see alsoAppendix 4.

Table 8
Effect Parameters

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.29

0.36

0.96

6.1

lower 95%-cl

 

0.33

0.89

5.9

upper 95%-cl

 

0.40

1.0

6.4

Yield

Value

0.29

0.44

0.57

0.95

lower 95%-cl

 

0.38

0.52

0.92

upper 95%-cl

 

0.49

0.62

0.98

cl – confidence limit.

       Experimental Conditions

Table 9shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C. Temperature remained within the limits prescribed by the study plan (21‑24°C, constant within ±1°C).

Table 9
pH Levels Recorded during the Final Test

Terephthaldehyde

TWA conc. (mg/L)

pH

t=0h

t=72h

Control

8.0

8.2

0.29

8.0

8.2

1.1

8.0

8.0

7.4

8.0

8.0

27

8.1

8.0

95

8.1

7.9

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
IIn conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Terephthaldehyde inhibited growth rate and yield of this fresh water algae species significantly at Time Weighted Average concentrations of 1.1 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 6.1 mg/L with a 95% confidence interval ranging from 5.9 to 6.4 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.95 mg/L with a 95% confidence interval ranging from 0.92 to 0.98 mg/L.
The 72h-NOEC for growth rate inhibition was 0.29 mg/L.
The 72h-NOEC for yield inhibition was 0.29 mg/L.
Executive summary:

The objective of the study was to evaluate Terephthaldehyde for its ability to generate toxic effects inRaphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20and EC50for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Terephthaldehyde tested was a light yellow powder with a purity of 99.6% and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.94, 3.1, 9.7, 30 and 97 mg/L at nominally 1.0, 3.2, 10, 32 and 100 mg/L, respectively. During the 72-hour exposure period, the initially measured concentration at nominally 1.0 mg/L had decreased below the Limit of Detection (i.e. LOD=0.0073 mg/L). All other concentrations were measured to be at 0.40 – 95% of initial concentrations at the end of the test. Based on these results, the Time Weighted Average (TWA) concentrations were calculated to be 0.29, 1.1, 7.4, 27 and 95 mg/L at nominally 1.0, 3.2, 10, 32 and 100 mg/L, respectively. These TWA concentrations were used to determine the effect parameters.

A dose-related increase of algal growth inhibition was observed at all concentrations tested at the end of the test, resulting in 83% growth rate inhibition and 99% yield inhibition at the highest test concentration at the end of the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on TWA concentrations) obtained in this study are summarized in the table below.

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.29

0.36

0.96

6.1

lower 95%-cl

 

0.33

0.89

5.9

upper 95%-cl

 

0.40

1.0

6.4

Yield

Value

0.29

0.44

0.57

0.95

lower 95%-cl

 

0.38

0.52

0.92

upper 95%-cl

 

0.49

0.62

0.98

cl – confidence limit.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Terephthaldehyde inhibited growth rate and yield of this fresh water algae species significantly at Time Weighted Average concentrations of 1.1 mg/L and higher.