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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:
January 25, 2002 - August 23, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test concentrations were verified by chemical analysis. Duplicate samples, of 50 ml, were taken from control and test stock solutions at 0 hours and from culture vessels at 72 hours (replicates pooled) for analysis. The samples were not filtered to remove algal cells before analysis.
Additional samples were taken from flasks containing the "no algae" cultures at 72 hours (replicates pooled).
Preliminary trials were conducted on the stability of the test material in aqueous solution. Sealed vessels containing a known concentration of Oxazolidinyl ethyl methacrylate (OXEMA) were analysed at time 0 and after 24 hours under exposure to both light and dark conditions. Results showed the parent material degraded under both light and dark conditions. This indicated that Oxazolidinyl ethyl methacrylate (OXEMA) was not stable in aqueous solution.
Vehicle:
yes
Remarks:
diluent water
Details on test solutions:
Method of preparation
The test substance was dissolved in diluent water to give an initial stock solution of 100 mg/l. Serial dilutions of this stock solution were prepared with diluent water to give nominal test concentrations of 1.0, 2.2, 4.6, 10, and 22 mg/l. Algal pre-culture was mixed with the control and each test concentration at a rate of 6.3 ml per 1000 ml of medium to give a starting cell density of approximately 10^4 cells/ml.
Additional flasks containing the test substance at a nominal concentration equivalent to 4.6 mg/l, but without the presence of algal cells, were also prepared in order to obtain information on the extent of adsorptionlabsorption of the test substance by the algal cells. Throughout this report these cultures will be referred to as the "no algae" test cultures.
The test material completely dispersed when added to the dilution medium and remained in solution throughout the duration of the study.
Test organisms (species):
other: Selenastrum capricornutum
Details on test organisms:
Name: Selenastrum capricornutum, Strain No. CCAP 278/4.
Source: Algal cultures were obtained from the Culture Collection of Algae & Protozoa, CEH, Cumbria, UK.
Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (8590 - 8680 lux) in an orbital incubator at 22-23 degrees C, to give an algal suspension in log phase growth, characterised by a cell density of 1.61 x 10^6 cells/ml.
Test type:
static
Water media type:
other: sterile nutrient medium
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 degrees C
pH:
7.4-8.3
Nominal and measured concentrations:
The following definitive test concentrations were selected following a range finding study with concentrations of 0.1, 1.0, 10, 100 and 1000 mg/l. The dose response seen during this range finding indicated that the ECS, was between 1.0 and 10 mg/l.
Nominal concentrations: 1.0, 2.2,4.6, 10 and 22 mg/l
Geometric 24 hour Time Weighted Average (TWA) concentrations: 0.22, 0.47, 1.0, 2.2 and 4.9 mg/l
Geometric mean measured concentrations (0 and 72 hour): 0.18, 0.27, 0.40, 0.79 and 4.9 mg/l*
* Expired concentrations at nominal test concentrations 1.0, 2.2 and 4.6 mg/l were below the limit ofdetection (LOD 0.035 mg/l). In these instances the value of 0.035 mg/l was used as the expired value to calculate the geometric mean measured concentration.
Nominal exposure concentrations quoted in this report refer to the test material as received; no allowance has been made for a purity of less than 100%.
Details on test conditions:
Experimental design
Five test concentrations were prepared in triplicate plus one untreated control with six replicates.

Culture conditions
Conical flasks (250 ml) each containing 100 ml of test or control culture were loosely stoppered and placed without conscious bias in a Gallenkamp illuminated orbital incubator. The cultures were incubated, without medium renewal, for 72 hours under continuous illumination of between 6760 - 7360 lux provided by fluorescent tubes. The temperature was maintained at 23 degrees C. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 130 cycles per minute.

MEASUREMENT OF GROWTH
Samples were taken at 0,24,48 and 72 hours and the cell densities determined by direct counting using a Coulter Multisizer II particle counter.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.71 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
not specified
Results with reference substance (positive control):
Measured concentrations ranged from 93 - 98% of their nominal values at 0 hours and 0 - 5.3% of nominal at 72 hours. Geometric mean measured concentrations ranged from 8 - 22 % of their nominal values in this study. Actual geometric mean measured test concentrations were 0.18, 0.27,0.40,0.79 and 4.9 mg/l.

Comparison of the measured concentrations of the test substance in samples of the "no algae" test cultures could not be determined as expired concentrations were not detectable by analysis. However, the preliminary stability tests indicated the test material was not stable in aqueous solution and therefore accounts for the losses experienced during the 72 hour exposure period.

The following values were derived from the data based on geometric mean measured concentrations (based on 0 and 72 hour analytical measurements):
EbC50 (72 hours): 0.71 mg/l (95% confidence limits 0.61 - 0.84 mg/l)
ErC50 (0 - 72 hours): 0.88 mg/l (95% confidence limits 0.40 - 2.0 mg/l)
"No-observed effect concentration" (NOEC): 0.40 mg/l

The following values were derived from the data based on nominal concentrations:
EbC50(72 hours): 9.0 mg/l (95% confidence limits 6.6 - 12 mg/l)
ErC50 (0 - 72 hours): 13 mg/l (95% confidence limits 12 - 13 mg/l)
"NO-observed effect concentration" (NOEC): 4.6 mg/l

The following values were derived from the data based on the geometric 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 24 hours:
EbC50 (72 hours): 2.1 mg/l (95% confidence limits 1.9 - 2.2 mg/l)
ErC50 (0 - 72 hours): 2.8 mg/l (95% confidence limits 2.6 - 3.1 mg/l)
"No-observed effect concentration" (NOEC): 1.0 mg/l

The following values were derived from the data based on the geometric 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 72 hours:
EbC50 (0 - 72 hours): 2.0 mg/l (95% confidence limits 1.4 - 2.8 mg/l)
ErC50 (0-72 hours) 2.8 mg/l (95% confidence limits 2.7 - 2.8 mg/l)
"No-observed effect concentration" (NOEC): 1.0 mg/l

The geometric 24 hour Time Weighted Average (TWA) EC50 values have been presented in response to the toxicity observed over the 72 hour exposure period and the preliminary stability test of Oxazolidinyl ethyl methacrylate (OXEMA) conducted in algal medium. From the stability data, conducted under study number RAS 2571023215, the majority of the parent material was shown to have degraded during the initial 24 hour period of the exposure phase. Therefore, it is considered that the inhibitory effect of Oxazolidinyl ethyl methacrylate (OXEMA) occurs during this initial 24 hour phase of the study.

The stability test consisted of samples analysed approximately every hour for five hours from a solution Oxazolidinyl ethyl methacrylate (OXEMA) to determine the rate at which it degrades in solution. After 4.7 hours, 42% of the parent material had degraded. Using the data from the final analysis occasion, 4.7 hours, the half life of Oxazolidinyl ethyl methacrylate (OXEMA) in algal medium can be extrapolated due to this linear response. Therefore the estimated time for 50% of the parent material to degrade is 5.6 hours. This half life value calculated over the initial 24 hour period of the definitive test predicts the amount of parent material remaining in solution is 5.3%. The 24 hour time point concentrations are then based on 5.3% of the initial measured concentrations. The 24 hour TWA concentrations are calculated based on the geometric mean of the initial measured concentrations and the
24 hour time point concentration.

The test was considered valid because cell concentrations in control cultures increased by a factor of at least 16 within 72 hours (mean cell density of controls at 0 and 72 hours: 1 x 10^4 and 1.4 x 10^6 cells/ml, respectively).
The pH of the control cultures was 7.7 at 0 hours and from 7.4 - 8.2 at 72 hours, and therefore remained constant to within 1 unit over the 72 hour test period.

All test and control cultures were inspected microscopically at 72 hours. No abnormalities were detected in any of the cultures examined.
No cultures showed any signs of contamination by foreign algal cells or bacteria.

Table 1. Cell densities for control and test cultures for algae exposed for 72 hours to OXEMA

Concentration (mg/l)

Geometric mean measured concentration (mg/l)

pH

Cell densities (cells/ml)

pH

0* hour

0# hour

24 hour

48 hour

72 hour

72 hour

Control

R1

ND

7.7

1000

64032

331760

1463100

7.4

R2

59100

321020

1498400

7.7

R3

62864

337080

1435700

8.2

R4

67728

327200

1335200

8.0

R5

68920

332940

1380400

7.7

R6

63560

314000

1414600

7.8

Mean

-

-

1000

64367

327333

1421233

-

1.0

R1

0.18

7.7

1000

70488

325720

1484500

8.3

R2

66996

325360

1398500

7.8

R3

69312

314620

1558100

7.7

Mean

-

-

1000

68932

321900

1480367

-

2.2

R1

0.27

7.8

1000

60576

329400

1468200

7.8

R2

68368

337820

1632600

7.7

R3

60548

331220

1456900

7.6

Mean

-

-

1000

63164

332813

1519233

-

4.6

R1

0.40

7.8

1000

63728

333320

1692600

7.8

R2

64492

305900

1418900

7.7

R3

67864

308620

1353100

7.7

Mean

-

-

1000

65361

315947

1488200

-

10

R1

0.79

7.8

1000

33245

109560

407800

7.6

R2

28405

96590

339680

7.7

R3

33348

98870

341520

7.6

Mean

-

-

1000

31666

101673

363000

-

22

R1

4.9

8.0

1000

14625

16849

15699

7.7

R2

14399

15350

14801

7.7

R3

16406

17384

14904

7.7

Mean

-

-

1000

15143

16528

15135

-

TemperatureC

-

22

23

23

23

23

21

Rn: Replicates

ND: none detected

-: not applicable

*: Measurements taken on stock solutions

#: for statistical purposes these have been assumed to be 1000 cells/ml based on algal stock

Table 2. Inhibition of growth of algae exposed for 72 hours to OXEMA

Nominal concentration (mg/l)

Geometric mean measured concentration (0 and 72 analytical measurements) (mg/l)#

Area under curve at 72 hours

% inhibition *

Growth rate (0-72 hours)

% inhibition *

Control

ND

26

-

0.069

-

1.0

0.18

27

<2.7>

0.069

<0.82>

2.2

0.27

27

<4.9>

0.070

<1.3>

4.6

0.40

26

<2.1>

0.069

<0.85>

10

0.79

7.0

73

0.050

28

22

4.9

0.34

99

0.0058

92

*: percentage inhibition values calculated using non-rounded data

ND: none detected

-: not sampled

< >: stimulated growth

#: geometric mean measured concentrations (0 and 72 analytical measurements) were used to calculate growth rate and biomass (AUC) inhibition

Table 3. OXEMA: Geometric mean measured concentrations

Nominal concentration (mg/l)

Number of samples analyzed

*Geometric mean measured concentration (0 and 72 analytical measurements) (mg/l)

**% nominal

*Geometric 24 hour Time Weighted Average (TWA) concentrations (mg/l)

**% nominal

Control

2

ND

-

ND

-

1.0

2

0.18#

18

0.22

22

2.2

2

0.27#

12

0.47

21

4.6

2

0.40#

9

1.0

22

10

2

0.79

8

2.2

22

22

2

4.9

22

4.9

22

ND: none detected

-: not applicable

#: calculated based on the limit of detection (0.035 mg/l) value for the expired samples

*: calculated on unrounded values

**: calculated on rounded values

Validity criteria fulfilled:
yes
Conclusions:
Oxazolidinyl ethyl methacrylate (OXEMA) inhibited the growth of Selenastrum capvicornutum, Strain No. CCAP 27814 at concentrations tested in excess of nominally 4.6 mg/l under the conditions of this test, equivalent to the geometric mean measured concentration (based on 0 and 72 hour analytical measurements) of 0.40 mg/l. This is also equivalent to the 24 hour Time Weighted Average (TWA) concentration of 1.0 mgl/.

The EbC50 (72 hours) was 9.0 mg/l based on nominal concentrations (equivalent to geometric mean measured concentration (based on 0 and 72 hour analytical measurements) of 0.71 mg/l).
The ErC50 (0 - 72 hours) was 13 mg/l based on nominal concentration (equivalent to geometric mean measured concentration (based on 0 and 72 hour analytical measurements) of 0.88 mg/l).
The EbC50 (72 hours) was 2.1 mg/l based on 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 24 hours.
The ErC50 (0 - 72 hours) was 2.8 mg/l based on 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 24 hours.
Executive summary:

A study was conducted to assess the inhibitory effect of Oxazolidinyl ethyl methacrylate (OXEMA) on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4. This study was conducted in accordance with the test guidelines EC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 and OECD Guideline for Testing of Chemicals No. 201.

Triplicate algal cultures, with an initial cell count of approximately 1 x l04 cells/ml, were exposed to Oxazolidinyl ethyl methacrylate (OXEMA) at nominal test concentrations of 1.0, 2.2, 4.6, 10 and 22 mg/l. Actual geometric mean measured test concentrations were 0.18, 0.27, 0.40, 0.79 and 4.9 mg/l. Measured concentrations ranged from 93 - 98% of their nominal values at 0 hours and from 0 - 5.3% of their values at 72 hour in this study. Stability tests indicated the test material was not stable in dilute aqueous solution and therefore accounts for the losses experienced after the 72 hour exposure period. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at 23°C for 72 hours. Cell numbers were counted daily to monitor growth.

Environmental conditions, light intensity and temperature, remained within the ranges specified by the protocol throughout duration of the study.

The following values were derived from the data based on geometric mean measured concentrations (based on 0 and 72 hour analytical measurements):

EbC50 (72 hours): 0.71 mg/l (95% confidence limits 0.61 - 0.84 mg/l)

ErC50 (0 - 72 hours): 0.88 mg/l (95% confidence limits 0.40 - 2.0 mg/l)

"No-observed effect concentration" (NOEC): 0.40 mg/l

The following values were derived from the data based on nominal concentrations:

EbC50(72 hours): 9.0 mg/l (95% confidence limits 6.6 - 12 mg/l)

ErC50 (0 - 72 hours): 13 mg/l (95% confidence limits 12 - 13 mg/l)

"NO-observed effect concentration" (NOEC): 4.6 mg/l

The following values were derived from the data based on the geometric 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 24 hours:

EbC50 (72 hours): 2.1 mg/l (95% confidence limits 1.9 - 2.2 mg/l)

ErC50 (0 - 72 hours): 2.8 mg/l (95% confidence limits 2.6 - 3.1 mg/l)

"No-observed effect concentration" (NOEC): 1.0 mg/l

The following values were derived from the data based on the geometric 24 hour Time Weighted Average (TWA) concentrations, and algal cell densities determined at 0 and 72 hours:

EbC50 (0 - 72 hours): 2.0 mg/l (95% confidence limits 1.4 - 2.8 mg/l)

ErC50 (0-72 hours) 2.8 mg/l (95% confidence limits 2.7 - 2.8 mg/l)

"No-observed effect concentration" (NOEC): 1.0 mg/l

EbC50 (“x” hours): The median effective concentration for inhibition of growth based on a comparison of areas under the growth curves after "x" hours

ErC50 (“x”-“y” hours): The median effective concentration for inhibition of growth based on a comparison of growth rates (from "x" to "y" hours)

Oxazolidinyl ethyl methacrylate (OXEMA) inhibited the growth of Selenastrum capricornutum, Strain No. CCAP 278/4 at concentrations tested in excess of nominally 4.6 mg/l under the conditions of this test, equivalent to the geometric mean measured concentration (based on 0 and 72 hour analytical measurements) of 0.40 mg/l. This is also equivalent to the 24 hour Time Weighted Average (TWA) concentration of 1.0 mg/l.

Description of key information

The key study is an OECD Guideline 201, EU Method C.3, GLP compliant study on green algae (Selenastrum capricornutum).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.88 mg/L
EC10 or NOEC for freshwater algae:
0.4 mg/L

Additional information

A study was conducted to assess the inhibitory effect of OXEMA on the growth of the unicellular green alga Selenastrum capricornutum. 

 

Triplicate algal cultures, with an initial cell count of approximately 1 x 106cells/ml, were exposed to OXEMA at nominal test concentrations of 1.0, 2.2, 4.6, 10 and 22 mg/l. Actual geometric mean measured test concentration were 0.18, 0.27, 0.40, 0.79 and 4.9 mg/l. Measured concentrations ranged from 93 – 98 % of their nominal values at 0 hours and from 0 –53% of their values at 72 hours. Stability tests indicated the test material was not stable in dilute aqueous solution and therefore accounts for the losses experienced after the 72 hour exposure period. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at 23º C for 72 hours. Cell numbers were counted daily to monitor growth.

 

The following values were derived from the data based on geometric mean measured concentrations (based on 0 and 72 hour analytical measurements).

 EbC50(72 hours): 0.71 mg/l  (95% confidence limits: 0.61 – 0.84 mg/l)

 ErC50(0 – 72 hours):  0.88 mg/l  (95% confidence limits: 0.40 – 2.0 mg/l)

 “No-observed effect concentration” (NOEC): 0.40 mg/l

 

The following values were derived from the data based on nominal concentrations:

 EbC50(72 hours): 9.0 mg/l  (95% confidence limits: 6.6 – 12 mg/l)

 ErC50(0 – 72 hours): 13 mg/l  (95% confidence limits: 12 – 13 mg/l)

 “No-observed effect concentration” (NOEC): 4.6 mg/l

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